ErbB4 receptor-specific neuregulin related ligands and uses therefor

ABSTRACT

The invention concerns a novel neuregulin related ligand (NRG3) including fragments and variants thereof, as new members of the neuregulin family of compounds. The invention also concerns methods and means for producing NRG3. The native polypeptides of the invention are characterized by containing an extracellular domain including an EGF-like domain, a transmembrane domain and a cytoplasmic domain. Isolated nucleotide sequences encoding such polypeptides, expression vectors containing the nucleotide sequences, recombinant host cells transformed with the vectors, and methods for the recombinant production for the novel NRG3s are also within the scope of the invention.

RELATED APPLICATION

This application is a non-provisional application filed under 37 CFR1.53(b)(1), claiming priority under 35 USC 119(e) to provisionalapplication No. 60\052,019 filed Jul. 9, 1997, the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention concerns novel neuregulin related ligands. Moreparticularly, the invention relates to a new member of the neuregulinfamily and functional derivatives of the novel polypeptide.

BACKGROUND OF THE INVENTION

Signal transduction affecting cell growth and differentiation isregulated in part by phosphorylation of various cellular proteins.Protein tyrosine kinases are enzymes that catalyze this process.Receptor protein tyrosine kinases are believed to direct cellular growthvia ligand-stimulated tyrosine phosphorylation of intracellularsubstrates. Growth factor receptor protein tyrosine kinases of the classI subfamily include the 170 kDa epidermal growth factor receptor (EGFR)encoded by the erbB1 gene. erbB1 has been causally implicated in humanmalignancy. In particular, increased expression of this gene has beenobserved in more aggressive carcinomas of the breast, bladder, lung andstomach. The second member of the class I subfamily, p185^(neu), wasoriginally identified as the product of the transforming gene fromneuroblastomas of chemically treated rats. The neu gene (also callederbB2 and HER2) encodes a 185 kDa receptor protein tyrosine kinase.Amplification and/or overexpression of the human HER2 gene correlateswith a poor prognosis in breast and ovarian cancers (Slamon et al.,(1987) Science 235:177-182; and Slamon et al., (1989) Science244:707-712). Overexpression of HER2 has been correlated with othercarcinomas including carcinomas of the stomach, endometrium, salivarygland, lung, kidney, colon and bladder. A further related gene, callederbB3 or HER3, has also been described (Kraus et al., (1989) Proc. Natl.Acad. Sci. USA 86:9193-9197). Kraus et al. (1989) discovered thatmarkedly elevated levels of erbB3 mRNA were present in certain humanmammary tumor cell lines indicating that erbB3, like erbB1 and erbB2,may play a role in human malignancies. The erbB3 gene has been found tobe overexpressed in breast (Lemoine et al. (1992) Br. J. Cancer66:1116-1121), gastrointestinal (Poller et al. (1992) J. Pathol.168:275-280, Rajkumer et al. (1993) J. Pathol. 170:271-278, and Sanidaset al. (1993) Int. J. Cancer 54:935-940, and pancreatic cancers (Lemoineet al. (1992) J. Pathol. 168:269-273, and Friess et al. (1995) ClinicalCancer Research 1:1413-1420).

The class I subfamily of growth factor receptor protein tyrosine kinaseshas been further extended to include the HER4/Erb4 receptor (EP PatAppln No 599,274; Plowman et al. (1993) Proc. Natl. Acad. Sci. USA90:1746-1750; and Plowman et al. (1993) Nature 366:473-475. Plowman etal. found that increased HER4 expression closely correlated with certaincarcinomas of epithelial origin, including breast adenocarcinomas.Diagnostic methods for detection of human neoplastic conditions(especially breast cancers) which evaluate HER4 expression are describedin EP Pat Appln No. 599,274.

The quest for the activator of the HER2 oncogene has lead to thediscovery of a family of polypeptides, collectively called neuregulins(NRG1). These proteins appear to result from alternate splicing of asingle gene which was mapped to the short arm of human chromosome 8 byOrr-Urtreger et al. (1993) Proc. Natl. Acad. Sci. USA 90:1867-1871.

Holmes et al. isolated and cloned a family of polypeptide activators forthe HER2 receptor which they called heregulin-α (HRG-α), heregulin-β1(HRG-β1), heregulin-β2 (HRG-β2), heregulin-β2-like (HRG-β2-like), andheregulin-β3 (HRG-β3). See Holmes et al. (1992) Science 256:1205-1210;WO 92/20798; and U.S. Pat. No. 5,367,060. The 45 kDa polypeptide, HRG-α,was purified from the conditioned medium of the MDA-MB-231 human breastcancer cell line. These researchers demonstrated the ability of thepurified heregulin polypeptides to activate tyrosine phosphorylation ofthe HER2 receptor in MCF7 breast tumor cells. Furthermore, the mitogenicactivity of the heregulin polypeptides on SK-BR-3 cells (which expresshigh levels of the HER2 receptor) was illustrated. Like other growthfactors which belong to the EGF family, soluble HRG polypeptides appearto be derived from a membrane bound precursor (called pro-HRG) which isproteolytically processed to release the 45 kDa soluble form. Thesepro-HRGs lack a N-terminal signal peptide.

While heregulins are substantially identical in the first 213 amino acidresidues, they are classified into two major types, α and β, based ontwo variant EGF-like domains which differ in their C-terminal portions.Nevertheless, these EGF-like domains are identical in the spacing of sixcysteine residues contained therein. Based on an amino acid sequencecomparison, Holmes et al. found that between the first and sixthcysteines in the EGF-like domain, HRGs were 45% similar toheparin-binding EGF-like growth factor (HB-EGF), 35% identical toamphiregulin (AR), 32% identical to TGF-α, and 27% identical to EGF.

The 44 kDa neu differentiation factor (NDF), which is the rat equivalentof human HRG, was first described by Peles et al. (1992) Cell69:205-216; and Wen et al. (1992) Cell 69:559-572. Like the HRGpolypeptides, NDF has an immunoglobulin (Ig) homology domain followed byan EGF-like domain and lacks a N-terminal signal peptide. Subsequently,Wen et al. (1994) Mol. Cell. Biol. 14(3):(1909-1919 carried out cloningexperiments to extend the family of NDFs. This work revealed sixdistinct fibroblastic pro-NDFs. Adopting the nomenclature of Holmes etal., the NDFs are classified as either α or β polypeptides based on thesequences of the EGF-like domains. Isoforms 1 to 4 are characterized onthe basis of the region between the EGF-like domain and transmembranedomain. Also, isoforms a, b and c are described which have variablelength cytoplasmic domains. These researchers conclude that differentNDF isoforms are generated by alternative splicing and perform distincttissue-specific functions. See also EP 505 148; WO 93/22424; and WO94/28133 concerning NDF.

Falls et al. (1993) Cell 72:801-815 describe another member of theheregulin family which they call acetylcholine receptor inducingactivity (ARIA) polypeptide. The chicken-derived ARIA polypeptidestimulates synthesis of muscle acetylcholine receptors. See also WO94/08007. ARIA is a β-type heregulin and lacks the entire spacer regionrich in glycosylation sites between the Ig-like domain and EGF-likedomain of HRGα, and HRGβ1-β3.

Marchionni et al. identified several bovine-derived proteins which theycall glial growth factors (GGFs) (Marchionni et al. (1993) Nature362:312-318). These GGFs share the Ig-like domain and EGF-like domainwith the other heregulin proteins described above, but also have anamino-terminal kringle domain. GGFs generally do not have the completeglycosylated spacer region between the Ig-like domain and EGF-likedomain. Only one of the GGFs, GGFII, possessed a N-terminal signalpeptide. See also WO 92/18627; WO 94/00140; WO 94/04560; WO 94/26298;and WO 95/32724 which refer to GGFs and uses thereof.

Ho et al. in (1995) J. Biol. Chem. 270(4):14523-14532 describe anothermember of the heregulin family called sensory and motor neuron-derivedfactor (SMDF). This protein has an EGF-like domain characteristic of allother heregulin polypeptides but a distinct N-terminal domain. The majorstructural difference between SMDF and the other heregulin polypeptidesis the lack in SMDF of the Ig-like domain and the “glyco” spacercharacteristic of all the other heregulin polypeptides. Another featureof SMDF is the presence of two stretches of hydrophobic amino acids nearthe N-terminus.

While the heregulin polypeptides were first identified based on theirability to activate the HER2 receptor (see Holmes et al., supra), it wasdiscovered that certain ovarian cells expressing neu and neu-transfectedfibroblasts did not bind or crosslink to NDF, nor did they respond toNDF to undergo tyrosine phosphorylation (Peles et al (1993) EMBO J.12:961-971). This indicated that another cellular component wasnecessary for conferring full heregulin responsiveness. Carraway et al.subsequently demonstrated that ¹²⁵I-rHRGβ1₁₇₇₋₂₄₄ bound to NIH-3T3fibroblasts stably transfected with bovine erbB3 but not tonon-transfected parental cells. Accordingly, they conclude that ErbB3 isa receptor for HRG and mediates phosphorylation of intrinsic tyrosineresidues as well as phosphorylation of ErbB2 receptor in cells whichexpress both receptors. Caraway et al. (1994) J. Biol. Chem.269(19):14303-14306. Sliwkowski et al. (1994) J. Biol. Chem.269(20):14661-14665 found that cells transfected with HER3 alone showlow affinities for heregulin, whereas cells transfected with both HER2and HER3 show higher affinities.

This observation correlates with the “receptor cross-talking” describedpreviously by Kokai et al., Cell 58:287-292 (1989); Stern et al. (1988)EMBO J. 7:995-1001; and King et al., 4:13-18 (1989). These researchersfound that binding of EGF to the EGFR resulted in activation of the EGFRkinase domain and cross-phosphorylation of p185^(HER2). This is believedto be a result of ligand-induced receptor heterodimerization and theconcomitant cross-phosphorylation of the receptors within theheterodimer (Wada et al. (1990) Cell 61:1339-1347).

Plowman and his colleagues have similarly studied p₁₈₅ ^(HER4)/p₁₈₅^(HER2) activation. They expressed p185^(HER2) alone, p185^(HER4) alone,or the two receptors together in human T lymphocytes and demonstratedthat heregulin is capable of stimulating tyrosine phosphorylation ofp185^(HER4), but could only stimulate p185^(HER2) phosphorylation incells expressing both receptors. Plowman et al., Nature 336:473-475(1993). Thus, heregulin is the only known example of a member of the EGFgrowth factor family that can interact with several receptors. Carrawayand Cantley (1994) Cell 78:5-8.

The biological role of heregulin has been investigated by severalgroups. For example, Falls et al., (discussed above) found that ARIAplays a role in myotube differentiation, namely affecting the synthesisand concentration of neurotransmitter receptors in the postsynapticmuscle cells of motor neurons. Corfas and Fischbach demonstrated thatARIA also increases the number of sodium channels in chick muscle.Corfas and Fischbach (1993) J. Neuroscience 13(5): 2118-2125. It hasalso been shown that GGFII is mitogenic for subconfluent quiescent humanmyoblasts and that differentiation of clonal human myoblasts in thecontinuous presence of GGFII results in greater numbers of myotubesafter six days of differentiation (Sklar et al. (1994) J. Cell Biochem.,Abst. W462, 18D, 540). See also WO 94/26298 published Nov. 24, 1994.

Holmes et al., supra, found that HRG exerted a mitogenic effect onmammary cell lines (such as SK-BR-3 and MCF-7). The mitogenic activityof GGFs on Schwann cells has also been reported. See, e.g., Brockes etal. (1980) J. Biol. Chem. 255(18):8374-8377; Lemke and Brockes (1984) J.Neurosci. 4:75-83; Brockes et al. (1984) J. Neuroscience 4(1):75-83;Brockes et al. (1986) Ann. Neurol. 20(30:317-322; Brockes, J. (1987)Methods in Enzym. 147:217-225 and Marchionni et al., supra. Schwanncells constitute important glial cells which provide myelin sheathingaround the axons of neurons, thereby forming individual nerve fibers.Thus, it is apparent that Schwann cells play an important role in thedevelopment, function and regeneration of peripheral nerves. Theimplications of this from a therapeutic standpoint have been addressedby Levi et al. (1994) J. Neuroscience 14(3):1309-1319. Levi et al.discuss the potential for construction of a cellular prosthesiscomprising human Schwann cells which could be transplanted into areas ofdamaged spinal cord. Methods for culturing Schwann cells ex vivo havebeen described. See WO 94/00140 and Li et al. (1996) J. Neuroscience16(6):2012-2019.

Pinkas-Kramarski et al. found that NDF seems to be expressed in neuronsand glial cells in embryonic and adult rat brain and primary cultures ofrat brain cells, and suggested that it may act as a survival andmaturation factor for astrocytes (Pinkas-Kramarski et al. (1994) PNAS,USA 91:9387-9391). Meyer and Birchmeier (1994) PNAS, USA 91:1064-1068analyzed expression of heregulin during mouse embryogenesis and in theperinatal animal using in situ hybridization and RNase protectionexperiments. These authors conclude that, based on expression of thismolecule, heregulin plays a role in vivo as a mesenchymal and neuronalfactor. Also, their findings imply that heregulin functions in thedevelopment of epithelia. Similarly, Danilenko et al. (1994) Abstract3101, FASEB 8(4-5):A535, found that the interaction of NDF and the HER2receptor is important in directing epidermal migration anddifferentiation during wound repair.

Although NRG1 was initially proposed to be the ligand for the receptortyrosine kinase ErbB2, further studies have demonstrated that activationof ErbB2 frequently occurred as a result of NRG1 binding to ErbB3(Sliwkowski, M. X., et al. (1994) J. Biol. Chem. 269:14661-14665) orErbB4 (Plowman, G. D. et al. (1993) Nature 366:473475; and Carraway, K.L. and Cantley, L. C. (1994) Cell 78:5-8) receptors. Recent studies havebegun to highlight the roles of NRG1, ErbB2 receptor and ErbB4 receptorin the development of the heart. Mice lacking ErbB4 receptor, ErbB2receptor or NRG1 die during mid-embryogenesis (embryonic day 10.5) fromthe aborted development of myocardial trabeculae in the ventricle (Meyer& Birchmeier (1995) Nature 378:386-90; Gassmann et al. (1995) Nature378:3904; and Lee et al. (1995) Nature 38:394-8). These results areconsistent with the view that NRG 1, expressed in the endocardium, is animportant ligand required for the activation of ErbB2 and ErbB4receptors in the myocardium.

These same studies suggest that NRG1 and ErbB2 receptor may play adifferent role than ErbB4 receptor in the development of the hind brain.NRG1 is expressed in the neuroepithelium and cells arising fromrhombomeres 2, 4 and 6, while ErbB4 receptor is expressed in rhombomeres3 and 5. NRG1 and ErbB2 receptor knockout mice exhibit a loss of cellsand axons of the cranial sensory ganglia. In contrast, ErbB4 receptordeficient mice do not exhibit a loss of cellularity in the cranialganglia. Rather, the organization, spacing and pattern of innervation ofthese ganglia to and from the central nervous system is disrupted(Gassmann et al., supra). One possible reason for this difference inhindbrain phenotypes of NRG1 and ErbB4 receptor knockout mice is thatadditional ligand(s) distinct from NRG1 may be recognized by ErbB4 inthe CNS (Gassmann et al., supra).

SUMMARY OF THE INVENTION

The present invention is based on the identification, recombinantproduction and characterization of a novel member of the family ofneuregulins (NRG1). More specifically, the invention concerns a novelpolypeptide, NRG3, comprising an EGF-like domain distinct from EGF-likedomains of NRG1 and NRG2. In addition, the NRG3 disclosed hereindisplays distinct receptor binding characteristics relative to otherneuregulin-like polypeptides.

In analyzing the homologous sequence motif, homology to the EGF-likedomain of NRG1 was observed in the subset of amino acids that areconserved in most neuregulins. Based upon this observation and theobserved ErbB4 receptor binding characteristics, the novel protein,NRG3, has been identified as a new member of the family of neuregulins.The novel protein contains domains that are distantly related to, butdistinct from, those found in the other members of the NRG1 family. Inaddition, it is expressed primarily in embryonic and adult tissues ofthe central nervous system. NRG3 represents a novel member of theneuregulin family of compounds, members of which are involved in cellproliferation and differentiation, epithelial development, cardiacdevelopment, neurological development, as well as acting as glial cellmitogens, and as mesenchymal and neuronal factors.

In one aspect, the present invention concerns a novel isolated mammalianNRG3 polypeptide having an EGF-like domain, and functional derivativesof the novel NRG3, which polypeptides bind the ErbB4 receptor. Thenative polypeptides within the scope of the present invention arecharacterized as containing an extracellular domain including anEGF-like domain, a transmembrane domain and a cytoplasmic domain. Thepresent invention specifically includes the soluble forms of the novelNRG3 ligand molecules of the invention, which have a transmembranedomain that cannot associate with a cell membrane, and optionally devoidof all or part of the cytoplasmic domain. By “transmembrane domain” ismeant a domain of the polypeptide that contains a sufficient number ofhydrophobic amino acids to allow the polypeptide to insert and anchor ina cell membrane. By “transmembrane domain that cannot associate with acell membrane” is meant a transmembrane domain that has been altered bymutation or deletion such that is insufficiently hydrophobic to allowinsertion or other association with a cell membrane. Such atransmembrane domain does not preclude, for example, the fusion of theNRG3 of the invention, or fragment thereof, with a secretion signalsequence useful for secretion of the polypeptide from the cell, aninsufficient number of hydrophobic amino acid side chains are presentdevoid of an active transmembrane domain does not insert into a cellmembrane. Mutations or alterations of the amino acid sequence useful toachieve an inactive transmembrane domain include, but are not limitedto, deletion or substitution of amino acids within the transmembranedomain.

In a particular embodiment, the invention concerns isolatedpolypeptides, preferably NRG3 ligands, having at least 75% amino acididentity to polypeptides selected from the group consisting of

-   -   (1) a polypeptide comprising the amino acid sequence encoding        the EGF-like domain shown in FIG. 3 (SEQ ID NO:4);    -   (2) a polypeptide comprising the amino acid sequence encoding        the extracellular domain of mouse or human NRG3 shown in FIG. 3        (SEQ ID NO: 3 or SEQ ID NO:7, respectively);    -   (3) a polypeptide comprising the amino acid sequence of the        native mouse or human NRG3 polypeptide shown in FIG. 3 (SEQ ID        NO: 2 and SEQ ID NO:6, respectfully);    -   (4) a further mammalian homologue of polypeptide (1)-(3);    -   (5) a soluble form of any of the polypeptides (1)-(4) devoid of        an active transmembrane domain; and    -   (6) a derivative of any of the polypeptides (1)-(5), retaining        the qualitative EGF-like domain and NRG3 receptor binding        properties of a polypeptide (1)-(5).

While the native NRG3 polypeptides of the present invention areglycoproteins, the present invention also encompasses variant moleculesunaccompanied by native glycosylation or having a variant glycosylationpattern. Preferably, the EGF-like domain of the NRG3 polypeptide isunglycosylated.

In a further embodiment, the invention includes an antagonist of a novelNRG3 of the present invention. The antagonist of the invention may be apeptide that binds an NRG3 such as an anti-NRG3 antibody or bindingfragment thereof. Preferably, the NRG3 antagonist of the inventionsubstantially reduces binding of a natural ErbB4 receptor ligand, suchas an NRG3, to the ErbB4 receptor, thereby preventing or limitingactivation of the receptor. In a preferred embodiment, the antagonistreduces NRG3 binding to its receptor to less than 50%, preferably lessthan 20%, most preferably less than 10% of the binding of an NRG3 underlike conditions.

In yet another embodiment, the invention includes an agonist of a novelNRG3 of the present invention. The agonist of the invention may be aNRG3, or it may be an anti-NRG3 receptor antibody or receptor bindingfragment. An agonist NRG3 of the invention may also be an polypeptideencoded by an alternatively spliced form of the native NRG3-encodinggene, preferably comprising the NRG3 EGF-like domain disclosed herein.In an embodiment of the agonist of the invention, the NRG3 agonist is ananti-ErbB4 receptor antibody, which antibody binds to and activates theErbB4 receptor. Preferably, the binding affinity of the agonist is atleast 25% of the affinity of the native ligand, more preferably at least50%, and most preferably at least 90% of the affinity of the nativeligand. Similarly, it is preferred that the agonist of the inventionactivate the ErbB4 receptor at the level of at least 25%, morepreferably at least 50%, most preferably at least 90% of activation ofthe native NRG3.

The invention further concerns a nucleic acid molecule encoding a novelNRG3 of the present invention, vectors containing such nucleic acid, andhost cells transformed with the vectors. The nucleic acid preferablyencodes at least the EGF-like domain of a native or variant ErbB4receptor-specific NRG3 of the present invention. The invention furtherincludes nucleic acids hybridizing under stringent conditions to thecomplement of a nucleic acid encoding a native ErbB4 receptor-specificNRG3 of the present invention, and encoding a protein retaining thequalitative ErbB4 receptor-specific binding properties of a native NRG3disclosed herein. In addition, the invention includes a nucleic aciddeposited with the American Type Culture Collection as ATCC 209156(pLXSN.mNRG3), which nucleic acid is an expression vector comprisingnucleic acid encoding the mouse NRG3 open reading frame (SEQ ID NO:1).The invention also includes a nucleic acid deposited with the AmericanType Culture Collection as ATCC 209157 (pRK5.tk.neo.hNRG3B1), whichnucleic acid is an expression vector comprising nucleic acid encoding ahuman NRG3 nucleic acid (SEQ ID NO:5). The invention also includes anucleic acid deposited with the American Type Culture Collection as ATCC209297 (pRK5.tk.neo.hNRG3B2), which nucleic acid is an expression vectorcomprising nucleic acid encoding an alternatively spliced form of humanNRG3 nucleic acid (SEQ ID NO:22) lacking nucleic acids 1585 to 1656 ofSEQ ID NO:5. The deduced amino acid sequence of the alternativelyspliced human NRG3B2 is found in SEQ ID NO:23 which lacks amino acids529 to 552 of SEQ ID NO:6. A comparison of the hNRG3B1 and hNRG3B2 aminoacid sequences is shown in FIG. 4B. The invention further includes NRG3amino acid sequences of mouse and human NRG3, alternatively splicedforms or fragments thereof, encoded by the deposited expression vectors.

In another aspect, the invention concerns a process for producing a NRG3of the invention, which process comprises transforming a host cell withnucleic acid encoding the desired NRG3, culturing the transformed hostcell and recovering the NRG3 produced from the host cell or host cellculture.

As an alternative to production of the NRG3 in a transformed host cell,the invention provides a method for producing NRG3 comprising: (a)transforming a cell containing an endogenous NRG3 gene with a homologousDNA comprising an amplifiable gene and a flanking sequence of at leastabout 150 base pairs that is homologous with a DNA sequence within or inproximity to the endogenous NRG3 gene, whereby the homologous DNAintegrates into the cell genome by recombination; (b) culturing the cellunder conditions that select for amplification of the amplifiable gene,whereby the NRG3 gene is also amplified; and thereafter (c) recoveringNRG3 from the cell.

In a further aspect, the invention concerns an antibody that bindsspecifically to a NRG3 of the present invention, and to a hybridoma cellline producing such an antibody.

In a still further aspect, the invention concerns an immunoadhesincomprising a novel NRG3 sequence, as disclosed herein, fused to animmunoglobulin sequence. The NRG3 sequence is preferably atransmembrane-domain-deleted form of a native or variant polypeptidefused to an immunoglobulin constant domain sequence, and comprises atleast the EGF-like domain of the extracellular domain of a native NRG3of the present invention. In another preferred embodiment, the NRG3sequence present in the immunoadhesin shows at least about 80% sequencehomology with the extracellular domain of the sequence shown in SEQ IDNO:3 NRG3 or SEQ ID NO:7 for mouse or human NRG3, respectively. Theimmunoglobulin constant domain sequence preferably is that of an IgG-1,IgG-2 or IgG-3 molecule, but may also be an IgA or IgM molecule.

In a further aspect, the invention encompasses a transgenic animalcomprising an altered NRG3 gene in which the polypeptide encoded by thealtered gene is not biologically active (non-functional), deleted, orhas no more than 70% wild type activity, preferably no more that 50%activity and more preferably no more than 25% activity of the nativeNRG3 polypeptide. In addition, a transgenic animal of the inventionincludes a transgenic animal comprising and expressing a native NRG3,alternatively spliced form of NRG3, or a fragment or variant thereof.Such transgenic animals are useful for the screening of potential NRG3agonists and antagonists.

The invention further concerns pharmaceutical compositions comprising aNRG3 as hereinabove defined in admixture with a pharmaceuticallyacceptable carrier. Dosages and administration of NRG3 in apharmaceutical composition may be determined by one of ordinary skill inthe art of clinical pharmacology or pharmacokinetics (see, for example,Mordenti, J. and Rescigno, A. (1992) Pharmaceutical Research 2:17-25;Morenti, J. et al. (1991) Pharmaceutical Research 8:1351-1359; andMordenti, J. and Chappell, W. (1989) “The use of interspecies scaling intoxicokinetics” in Toxicokinetics and New Drug Development, Yacobi etal. (eds), Pergamon Press, NY, pp. 42-96, each of which references areherein incorporated by reference in its entirety).

In an aspect of the invention, the isolated nucleic acid encoding theNRG3 of the invention, or fragment thereof, may also be used for in vivoor ex vivo gene therapy.

In an embodiment of the invention, a nucleic acid sequence encoding anNRG3, or fragment or variant thereof, is introduced into a cell of ananimal as part of an expression cassette such that the NRG3-encodingnucleic acid sequence is expressed in the cell. Preferably, the NRG3encoding nucleic acid sequence comprises sequences (such as a promotorsequence) for the control of NRG3 expression within the cell.Preferably, the expression cassette comprises a retroviral vector fordelivery of the nucleic acid sequence to a cell of the animal.

In a further embodiment of the invention, a host cell expressing an NRG3or NRG3 agonist is introduced into an animal, preferably a human, suchthat NRG3 or NRG3 agonist produced by the host cell is effective intreating a disorder responsive to increased local or systemic NRG3administration. Cells genetically engineered to express an NRG3,fragment or variant thereof, can be implanted in the host to provideeffective levels of factor or factors. The cells can be prepared,encapsulated, and implanted as provided in U.S. Pat. Nos. 4,892,538, and5,011,472, WO 92/19195, WO 95/05452, or Aebischer et al. (1996) NatureMedicine 2:696-699, for example, which references are hereinincorporated by reference in their entirety.

The present invention includes methods of enhancing survival,proliferation or differentiation of cells comprising the ErbB4 receptorin vivo and in vitro. Normally, the cells will be treated with the NRG3polypeptide or fragment or variant thereof. However, gene therapyapproaches have been described in the art and are encompassed by thepresent invention. These techniques include gene delivery to a cellusing adenovirus, herpes simplex I virus or adeno-associated virus aswell as lipid-based delivery systems (e.g. liposomes). Retroviruses areuseful for ex vivo gene therapy approaches. Accordingly, it is possibleto administer the nucleic acid encoding NRG3, resulting in expression ofthe NRG3 polypeptide, fragment or variant in the patient or in tissueculture. For exemplary gene therapy techniques see WO 93/25673 and thereferences cited therein.

An aspect of the invention is a method of treating a disorder byadministering to a mammal a cell encoding an NRG3 or fragment thereof,or agonist or antagonist of the NRG3 as necessary to treat the disorder,which cell secretes the NRG3 of the invention. An embodiment of theinvention is a method for preventing or treating damage to a nerve ordamage to other NRG3-expressing or NRG3-responsive cells, e.g. brain,heart, or kidney cells, which method comprises implanting cells thatsecrete NRG3, or fragment or agonist thereof, or antagonist as may berequired for the particular condition, into the body of patients in needthereof.

A further embodiment of the invention includes an implantation device,for preventing or treating nerve damage or damage to other cells astaught herein, containing a semipermeable membrane and a cell thatsecretes NRG3, or fragment or agonist thereof, (or antagonist as may berequired for the particular condition) encapsulated within the membrane,the membrane being permeable to NRG3, or fragment agonist thereof, andimpermeable to factors from the patient detrimental to the cells. Thepatient's own cells, transformed to produce NRG3 ex vivo, could beimplanted directly into the patient, optionally without suchencapsulation. The methodology for the membrane encapsulation of livingcells is familiar to those of ordinary skill in the art, and thepreparation of the encapsulated cells and their implantation in patientsmay be accomplished readily as is known in the art.

In accordance with the in vitro methods of the invention, cellscomprising the ErbB4 receptor are placed in a cell culture medium.Examples of ErbB4-receptor-containing cells include neural cells, e.g.,brain cells (such as neurons of the neocortex, cerebellum andhippocampus); cardiac cells; skeletal and smooth muscle cells; andcultured cells transformed with a recombinant NRG3.

Suitable tissue culture media are well known to persons skilled in theart and include, but are not limited to, Minimal Essential Medium (MEM),RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM). These tissueculture medias are commercially available from Sigma Chemical Company(St. Louis, Mo.) and GIBCO (Grand Island, N.Y.). The cells are thencultured in the cell culture medium under conditions sufficient for thecells to remain viable and grow in the presence of an effective amountof NRG3. The cells can be cultured in a variety of ways, includingculturing in a clot, agar, or liquid culture.

The cells are cultured at a physiologically acceptable temperature suchas 37° C., for example, in the presence of an effective amount of NRG3,fragment or variant. The amount of NRG3 may vary, but preferably is inthe range of about 0.1 ng/ml to about 1 mg/ml preferably about 0.1 ng/mlto about 0.1 ng/ml. The NRG3 can of course be added to the culture at adose determined empirically by those in the art without undueexperimentation. The concentration of NRG3 in the culture will depend onvarious factors, such as the conditions under which the cells and NRG3are cultured. The specific temperature and duration of incubation, aswell as other culture conditions, can be varied depending on suchfactors as, e.g., the concentration of the NRG3, and the type of cellsand medium. Those skilled in the art will be able to determine operativeand optimal culture conditions without undue experimentation.Proliferation, differentiation and/or survival of the cells (e.g.neurons) in the cultures can be determined by various assays known inthe art such as those described above.

It is contemplated that using NRG3 to enhance cell survival, growthand/or differentiation in vitro will be useful in a variety of ways. Forinstance, neural cells cultured in vitro in the presence of NRG3 can beinfused into a mammal suffering from reduced levels of the cells. Stablein vitro cultures can also be used for isolating cell-specific factorsand for expression of endogenous or recombinantly introduced proteins inthe cell. NRG3, fragments or variants thereof may also be used toenhance cell survival, proliferation and/or differentiation of cellswhich support the growth and/or differentiation of other cells in cellculture.

The invention also provides in vivo uses for NRG3. Based on the neuronalcell expression pattern of NRG3, it is believed that this molecule willbe particularly useful for treating diseases which involve neural cellgrowth such as demyelination, or damage or loss of glial cells (e.g.multiple sclerosis).

The invention further provides a method for treating a mammal comprisingadministering a therapeutically effective amount of NRG3, NRG3 fragment,or NRG3 agonist to the mammal. For example, the mammal may be sufferingfrom a neurological or muscular disorder. Where the disorder is aneurological disorder, NRG3 is believed to be useful in promoting thedevelopment, maintenance, and/or regeneration of neurons in vivo,including central (brain and spinal chord), peripheral (sympathetic,parasympathetic, sensory, and enteric neurons), and motoneurons.Accordingly, NRG3 may be utilized in methods for the diagnosis and/ortreatment of a variety of neurologic diseases or disorders which affectthe nervous system of a mammal, such as a human.

Such diseases or disorders may arise in a patient in whom the nervoussystem has been damaged by, e.g., trauma, surgery, stroke, ischemia,infection, metabolic disease, nutritional deficiency, malignancy, ortoxic agents. The agent is designed to promote the survival or growth ofneurons. For example, NRG3 can be used to promote the survival or growthof motoneurons that are damaged by trauma or surgery. Also, NRG3 can beused to treat motoneuron disorders, such as amyotrophic lateralsclerosis (Lou Gehrig's disease), Bell's palsy, and various conditionsinvolving spinal muscular atrophy, or paralysis. NRG3 can be used totreat human “neurodegenerative disorders”, such as Alzheimer's disease,Parkinson's disease, epilepsy, multiple sclerosis, Huntington's chorea,Down's Syndrome, nerve deafness, and Meniere's disease.

Further, NRG3 can be used to treat neuropathy, and especially peripheralneuropathy. “Peripheral neuropathy” refers to a disorder affecting theperipheral nervous system, most often manifested as one or a combinationof motor, sensory, sensorimotor, or autonomic neural dysfunction. Thewide variety of morphologies exhibited by peripheral neuropathies caneach be attributed uniquely to an equally wide number of causes. Forexample, peripheral neuropathies can be genetically acquired, can resultfrom a systemic disease, or can be induced by a toxic agent. Examplesinclude but are not limited to distal sensorimotor neuropathy, orautonomic neuropathies such as reduced motility of the gastrointestinaltract or atony of the urinary bladder. Examples of neuropathiesassociated with systemic disease include post-polio syndrome; examplesof hereditary neuropathies include Charcot-Marie-Tooth disease, Refsum'sdisease, Abetalipoproteinemia, Tangier disease, Krabbe's disease,Metachromatic leukodystrophy, Fabry's disease, and Dejerine-Sottassyndrome; and examples of neuropathies caused by a toxic agent includethose caused by treatment with a chemotherapeutic agent such asvincristine, cisplatin, methotrexate, or 3′-azido-3′-deoxythymidine.

The invention further provides a method for treating a mammal comprisingadministering a therapeutically effective amount of a NRG3 antagonist tothe mammal. The mammal in this latter case is one which could benefitfrom a reduction in NRG3 levels/biological activity.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the structure, synthesis, and usage as more fullyset forth below. Each reference cited herein is herein incorporated byreference in its entirety with particular attention to the descriptionof subject matter associated with the context of the citation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleic acid coding sequence of mouse NRG3 cDNA (mNRG3,SEQ ID NO:1) in which the start (ATG) and stop (TGA) codons of thecoding sequence are indicated by underlining.

FIG. 2 shows the nucleic acid coding sequence of human NRG3 cDNA(hNRG3B1, SEQ ID NO:5) in which the start (ATG) and stop (TGA) codons ofthe coding sequence are indicated by underlining.

FIG. 3 shows the nucleic acid coding sequence of an alternativelyspliced form of human NRG3 cDNA (hNRG3B2; SEQ ID NO:22) in which thestart (ATG) and stop (TGA) codons of the coding sequence are indicatedby underlining.

FIGS. 4A-4B. FIG. 4A shows the deduced amino acid sequences from mouse(mNRG3) and human (hNRG3B1) cDNA as shown in FIGS. 1 and 2. Mouse NRG3deduced amino acid sequence is depicted by SEQ ID NO:2 and human NRG3B1deduced amino acid sequence is depicted by SEQ ID NO:6. Various putativedomains within the amino acid sequences are shown. The EGF-like domain,the N-terminal hydrophobic segment (double underline), theserine/threonine-rich portion, and a predicted transmembrane domain(single underline) are highlighted. FIG. 4B shows the deduced amino acidsequences from hNRG3B1 and hNRG3B2 cDNA as shown in FIGS. 2 and 3. HumanNRGB1 deduced amino acid sequence is depicted by SEQ ID NO:6 and humanNRG3B2 deduced amino acid sequence is depicted by SEQ ID NO:23. Theregion of the NRG3 amino acid sequence that differs between the twohuman sequences is illustrated.

FIG. 5 shows a sequence alignment of the EGF-like domains of humanNRG3B1 (hNRG3.egf; SEQ ID NO:4); chicken ARIA (cARIA.egf; SEQ ID NO:9);human amphiregulin (hAR.egf; SEQ ID NO:10); human betacellulin(hBTC.egf; SEQ ID NO:11); human EGF (hEGF.egf; SEQ ID NO:12); humanheparin-binding EGF-like growth factor (hHB-EGF.egf; SEQ ID NO:13);human heregulin-α (hHRGα; SEQ ID NO:14); human heregulin-β(hHRGβ,.egf;SEQ ID NO:15); human TGF-α (hTGFα.egf; SEQ ID NO:16); and mouseepiregulin (mEPR.egf; SEQ ID NO:17). The sequences were analyzed usingSequence Analysis Programs, Genentech, Inc.

FIG. 6A-6H are FACS plots demonstrating binding of NRG3^(EGF).Fc toErbB4 receptor expressed on the surface of cells. In FIGS. 6A-6D,parental K562 cells (FIG. 6A) or K562 cells expressing either ErbB2receptor (K562^(erbB2) cells; FIG. 6B), ErbB3 receptor (K562^(erbB3)cells; FIG. 6C) or ErbB4 receptor (K₅₆₂ ^(erbB4) cells; FIG. 6D) wereexamined for the expression of corresponding receptors. Cells wereincubated with anti-ErbB2 receptor, anti-ErbB3 receptor or anti-ErbB4receptor antibodies as indicated before PE-conjugated secondary antibodywas added. “LOG PE” represents relative fluorescent intensity and“Counts” represents cell numbers. In FIGS. 6E-6H, NRG3^(EGF).Fc is shownby FACS analysis to bind to ErbB4 receptor expressing cells. ParentalK562 cells (FIG. 6E), K562^(erbB2) cells (FIG. 6F), K562^(erbB3) cells(FIG. 6G) and K562^(erbB4) cells (FIG. 6H) were incubated with orwithout NRG3^(EGF).Fc (containing gD tag) for 1 hour, followed byanti-gD-tag primary antibody and PE-conjugated secondary antibody.

FIG. 7 is a graphical analysis showing competitive inhibition of¹²⁵I-NRG3^(EGF).Fc binding to immobilized soluble ErbB4 receptor byNRG3^(EGF).Fc or NRG^(EGF). Soluble ErbB4 receptor was immobilized on96-well plates, and was incubated with various concentrations ofunlabeled NRG3^(EGF).Fc or NRG^(EGF) and constant amount of ¹²⁵I-labeledNRG3^(EGF).Fc for 1.5 hour at room temperature. The fraction ofradioactivity bound over total ¹²⁵I-NRG3^(EGF).Fc input is plottedagainst the concentration of competitor. Data of a representativeexperiment from four independent assays is shown. Error bars indicatestandard deviation of quadruplicate samples.

Before the present polypeptides, nucleic acids, vectors, and host cellsand processes for making such are described, it is to be understood thatthis invention is not limited to the particular compositions of matterand processes described, as such compounds and methods may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims.

DESCRIPTION OF THE EMBODIMENTS

Definitions

The phrases “novel neuregulin related ligand”, “novel NRG3”, “novelErbB4 receptor-specific NRG3” are used interchangeably and refer to anew member of the family of neuregulins, which NRG3 is expressedspecifically in the brain and nervous system of the embryo and adults,and to functional derivatives of such native polypeptides.

The term “NRG3” or “neuregulin related ligand” is defined herein to beany polypeptide sequence that possesses at least one biological property(as defined below) of native amino acid sequence NRG3 of SEQ ID NO:2 or6 (mouse or human, respectively) and additionally includes analternatively spliced form of human NRG3 having the amino acid sequenceof SEQ ID NO:23. This definition encompasses not only the polypeptideisolated from a native NRG3 source such as human MDA-MB-175 cells orfrom another source, such as another animal species or alternativelyspliced forms of NRG3, but also the polypeptide prepared by recombinantor synthetic methods. It also includes variant forms includingfunctional derivatives, allelic variants, naturally occurring isoformsand analogues thereof. Sometimes the NRG3 is “native NRG3” which refersto endogenous NRG3 polypeptide which has been isolated from a mammal.The NRG3 can also be “native sequence NRG3” insofar as it has the sameamino acid sequence as a native NRG3 (e.g. mouse (SEQ ID NO:2) or human(SEQ ID NO:6 or SEQ ID NO:23) NRG3 shown in FIGS. 4A and 4B). However,“native sequence NRG3” encompasses the polypeptide produced byrecombinant or synthetic means. “Mature NRG3” is soluble or secretedNRG3 released from the cell (i.e. lacking an N-terminal hydrophobicsequence). In this context, NRG3 refers to novel NRG3s comprising anEGF-like domain within an extracellular domain, a transmembrane domainand a cytoplasmic domain, with or without a native signal sequence, andnaturally occurring soluble forms of such NRG3s, with or without theinitiating methionine, whether purified from native source, synthesized,produced by recombinant DNA technology or by any combination of theseand/or other methods. The native NRG3s of the present inventionspecifically include the murine NRG3, the amino acid sequence of whichis shown in FIG. 4 (SEQ. ID. NO:2), and the human NRG3s having the aminoacid sequences shown in FIG. 4 (SEQ. ID. NO:6 or SEQ ID NO:23), andfragments or mammalian homologues or alternatively spliced forms ofthese native ligands. The novel native murine and human NRG3s of thepresent invention are about 713 and 720 amino acids in length,respectively, and comprise an EGF-like domain, the N-terminalhydrophobic segment, the serine/threonine-rich portion, a predictedtransmembrane domain, and a predicted intracellular domain. Theboundaries of these domain are indicated in FIG. 4 for the novel murineand human NRG3 sequences.

Optionally, the NRG3 is not associated with native glycosylation.“Native glycosylation” refers to the carbohydrate moieties which arecovalently attached to native NRG3 when it is produced in the mammaliancell from which the native NRG3 is derived. Accordingly, human NRG3produced in a non-human could be described as not being associated withnative glycosylation, for example it may be glycosylated other than thenative glycosylation. Sometimes, the NRG3 is not associated with anyglycosylation whatsoever (e.g. as a result of being producedrecombinantly in a prokaryote).

The term “EGF-like domain” refers to an extracellular epidermal growthfactor (EGF)-like domain of a polypeptide, preferably a NRG3 polypeptideof the invention. The EGF-like domain is sufficient to bind neuregulinreceptors and stimulate cellular responses (Holmes, W. E., et al. (1992)Science 256:1205-1210). Preferably, an EGF-like domain of the NRG3 ofthe invention has the amino acid sequence of the NRG3s shown in SEQ IDNO:4 (mouse or human NRG3 EGF-like domain), where the EGF-like domain isfrom about amino acid 284 to about amino acid 332 of human NRG3, andfrom about amino acid 286 to about amino acid 334 of mouse NRG3. TheNRG3 of the invention encompasses a polypeptide encoded by analternatively spliced form the NRG3 encoding gene, which alternativelyspliced form comprises the NRG3 EGF-like domain.

The term “ErbB” when used herein refers to any one or more of themammalian ErbB receptors (i.e. ErbB1 or epidermal growth factor (EGF)receptor; ErbB2 or HER2 receptor; ErbB3 or HER3 receptor; ErbB4 or HER4receptor; and any other member(s) of this class I tyrosine kinase familyto be identified in the future) and “erbB” refers to the mammalian erbBgenes encoding these receptors.

The terms “soluble form”, “soluble receptor”, “soluble NRG3”, “solubleNRG3”, and grammatical variants thereof, refer to variants of the nativeor variant NRG3s of the present invention which are devoid of afunctional transmembrane domain. In the soluble receptors thetransmembrane domain may be deleted, truncated or otherwise inactivatedsuch that they are not capable of cell membrane anchorage. If desired,such soluble forms of the NRG3s of the present invention mightadditionally have their cytoplasmic domains fully or partially deletedor otherwise inactivated.

A “functional derivative” of a polypeptide is a compound having aqualitative biological activity in common with the native polypeptide.Thus, a functional derivative of a native novel NRG3 of the presentinvention is a compound that has a qualitative biological activity incommon with such native NRG3. “Functional derivatives” include, but arenot limited to, fragments of native polypeptides from any animal species(including humans), derivatives of native (human and non-human)polypeptides and their fragments, and peptide and non-peptide analogs ofnative polypeptides, provided that they have a biological activity incommon with a respective native polypeptide.

As used herein, the term “fragments” refers to regions within thesequence of a mature native polypeptide. Preferably NRG3 fragments willhave a consecutive sequence of at least 20, and more preferably at least50, amino acid residues of the EGF-like domain of NRG3. The preferredfragments have about 30-150 amino acid residues which are identical to aportion of the sequence of NRG3 in SEQ ID NO:2 (from mouse), or in SEQID NO:6 or SEQ ID NO:23 (from human). The term “derivative” is used todefine amino acid sequence and glycosylation variants, and covalentmodifications of a native polypeptide. “Non-peptide analogs” are organiccompounds which display substantially the same surface as peptideanalogs of the native polypeptides. Thus, the non-peptide analogs of thenative novel NRG3s of the present invention are organic compounds whichdisplay substantially the same surface as peptide analogs of the nativeNRG3s. Such compounds interact with other molecules in a similar fashionas the peptide analogs, and mimic a biological activity of a native NRG3of the present invention. Preferably, amino acid sequence variants ofthe present invention retain at least one domain of a native NRG3,preferably an EGF-like domain, or have at least about 60% amino acidsequence identity, more preferably at least about 75% amino acidsequence identity, and most preferably at least about 90% amino acidsequence identity with a domain of a native NRG3 of the presentinvention. The amino acid sequence variants preferably show the highestdegree of amino acid sequence homology with the EGF-like domain ofnative NRG3s of the present invention. These are the domains which showthe highest percentage amino acid conservation between the novel NRG3sof the present invention and other members of the NRG3 family (see FIG.4).

The terms “isolated” or “substantially pure” refer to a polypeptide ornucleic acid which is free of other polypeptides or nucleic acids aswell as lipids, carbohydrates or other materials with which it isnaturally associated. An exception is made for glycosylation whereinsugar moieties are covalently attached to amino acids of the NRG3polypeptide of the invention. One of ordinary skill in the art canpurify a NRG3 polypeptide or nucleic acid encoding the polypeptide usingstandard techniques appropriate for each type of molecule.

The term “percent amino acid sequence identity” with respect to the NRG3sequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical with the residues in the NRG3sequence having the deduced amino acid sequence described in FIG. 1,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. N-terminal,C-terminal, or internal extensions, deletions, or insertions into theNRG3 sequence shall be construed as affecting sequence identity orhomology.

Another type of NRG3 variant is “chimeric NRG3”, which term encompassesa polypeptide comprising full-length NRG3 or a fragment thereof fused orbonded to a heterologous polypeptide. The chimera will normally share atleast one biological property with NRG3. Examples of chimeric NRG3sinclude immunoadhesins and epitope tagged NRG3. In another embodiment,the heterologous polypeptide is thioredoxin, a salvage receptor bindingepitope, cytotoxic polypeptide or enzyme (e.g., one which converts aprodrug to an active drug).

The terms “covalent modification” and “covalent derivatives” are usedinterchangeably and include, but are not limited to, modifications of anative polypeptide or a fragment thereof with an organic proteinaceousor non-proteinaceous derivatizing agent, fusions to heterologouspolypeptide sequences, and post-translational modifications. Covalentmodifications are traditionally introduced by reacting targeted aminoacid residues with an organic derivatizing agent that is capable ofreacting with selected sides or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. Certain post-translational modifications are theresult of the action of recombinant host cells on the expressedpolypeptide. Glutaminyl and asparaginyl residues are frequentlypost-translationally deamidated to the corresponding glutamyl andaspartyl residues. Alternatively, these residues are deamidated undermildly acidic conditions. Other post-translational modifications includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl, tyrosyl or threonyl residues, methylation of the α-aminogroups of lysine, arginine, and histidine side chains (T. E. Creighton(1983) Proteins: Structure and Molecular Properties, W.H. Freeman & Co.,San Francisco, pp. 79-86). Covalent derivatives/modificationsspecifically include fusion proteins comprising native NRG3 sequences ofthe present invention and their amino acid sequence variants, such asimmunoadhesins, and N-terminal fusions to heterologous signal sequences.

The term “biological activity” in the context of the present inventionis defined as the possession of at least one adhesive, regulatory oreffector function qualitatively in common with a native polypeptide.Preferred functional derivatives within the scope of the presentinvention are unified by retaining an EGF-like domain and ErbB4receptor-specific binding of a native NRG3 of the present invention.

The phrase “activating an ErbB receptor” refers to the act of causingthe intracellular kinase domain of an ErbB receptor to phosphorylatetyrosine residues. Generally, this will involve binding of NRG3 to anErbB4 receptor or ErbB4 receptor homodimer, which binding activates akinase domain of one or more of the receptors and thereby results inphosphorylation of tyrosine residues in one or more of the receptors,and/or phosphorylation of tyrosine residues in additional substratepolypeptide(s). ErbB receptor phosphorylation can be quantified usingthe tyrosine phosphorylation assays described below. It is understoodthat the NRG3 of the invention may itself be activated by interactionwith an ErbB receptor via the intracellular domain of NRG3. Thus, anNRG3-activating ligand that binds to the NRG3 (preferably binding to theextracellular domain, more preferably the EGF-like domain) includes, butis not limited to, a ligand, an antibody, or a receptor. Activation ofthe NRG3 may be through phosphorylation of the intracellular domain orother like event common to receptor/ligand mediated cell signaling. As amediator of cell signaling, the NRG3 of the invention is expected to beassociated with apoptosis, metabolic signaling, differentiation or cellproliferation.

“Identity” or “homology” with respect to a native polypeptide and itsfunctional derivative is defined herein as the percentage of amino acidresidues in the candidate sequence that are identical with the residuesof a corresponding native polypeptide, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. Neither N- or C-terminal terminal extensions norinsertions shall be construed as reducing identity or homology. Methodsand computer programs for the alignment are well known in the art. Forexample, the sequences disclosed herein were analyzed using SequenceAnalysis Programs, Genentech, Inc, Inc.

The term “agonist” is used to refer to peptide and non-peptide analogsof the native NRG3s of the present invention and to antibodiesspecifically binding such native NRG3s provided that they retain atleast one biological activity of a native NRG3. Preferably, the agonistsof the present invention retain the qualitative EGF-like domain bindingrecognition properties of the native NRG3 polypeptides.

The term “antagonist” is used to refer to a molecule inhibiting abiological activity of a native NRG3 of the present invention.Preferably, the antagonists herein inhibit the binding of a native NRG3of the present invention. Preferred antagonists essentially completelyblock the binding of a native NRG3 to an ErbB4 receptor to which itotherwise binds. A NRG3 “antagonist” is a molecule which prevents, orinterferes with, a NRG3 effector function (e.g. a molecule whichprevents or interferes with binding and/or activation of an ErbB4receptor by NRG3). Such molecules can be screened for their ability tocompetitively inhibit ErbB receptor activation by NRG3 in the tyrosinephosphorylation assay disclosed herein, for example. Preferredantagonists are those which do not substantially interfere with theinteraction of other heregulin polypeptides with ErbB receptor(s).Examples of NRG3 antagonists include neutralizing antibodies againstNRG3 and antisense polynucleotides against the NRG3 gene.

Ordinarily, the terms “amino acid” and “amino acids” refer to allnaturally occurring L-α-amino acids. In some embodiments, however,D-amino acids may be present in the polypeptides or peptides of thepresent invention in order to facilitate conformational restriction. Forexample, in order to facilitate disulfide bond formation and stability,a D amino acid cysteine may be provided at one or both termini of apeptide functional derivative or peptide antagonist of the native NRG3sof the present invention. The amino acids are identified by either thesingle-letter or three-letter designations: Asp D aspartic acid Ile Iisoleucine Thr T threonine Leu L leucine Ser S serine Tyr Y tyrosine GluB glutamic acid Phe F phenylalanine Pro P proline His H histidine Gly Gglycine Lys K lysine Ala A alanine Arg R arginine Cys C cysteine Trp Wtryptophan Val V valine Gln Q glutamine Met M methionine Asn Nasparagine

The term “amino acid sequence variant” refers to molecules with somedifferences in their amino acid sequences as compared to a native aminoacid sequence.

Substitutional variants are those that have at least one amino acidresidue in a native sequence removed and a different amino acid insertedin its place at the same position.

Insertional variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative sequence. Immediately adjacent to an amino acid means connectedto either the α-carboxy or α-amino functional group of the amino acid.

Deletional variants are those with one or more amino acids in the nativeamino acid sequence removed.

“Antibodies (Abs)” and “immunoglobulins (Igs)” are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 Daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one and (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light and heavy chain variable domains (Clothia et al.(1985) J. Mol. Biol. 186, 651-663; Novotny and Haber (1985) Proc. Natl.Acad. Sci. USA 82:4592-4596).

The light chains of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, delta, epsilon, γ, and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

The term “antibody” is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist and antagonistantibodies), antibody compositions with polyepitopic specificity, aswell as antibody fragments (e.g., Fab, F(ab′)₂, and Fv), so long as theyexhibit the desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein(1975) Nature 256:495, or may be made by recombinant DNA methods (see,e.g. U.S. Pat. No. 4,816,567 (Cabilly et al.) and Mage and Lamoyi (1987)in Monoclonal Antibody Production Techniques and Applications, pp.79-97, Marcel Dekker, Inc., New York). The monoclonal antibodies mayalso be isolated from phage libraries generated using the techniquesdescribed in McCafferty et al. (1990) Nature 348:552-554, for example.

“Humanized” forms of non-human (e.g. murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from thecomplementarity determining regions (CDRs) of the recipient antibody arereplaced by residues from the CDRs of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human FR residues. Furthermore, the humanized antibody may compriseresidues which are found neither in the recipient antibody nor in theimported CDR or FR sequences. These modifications are made to furtherrefine and optimize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. For further details see: Jones et al.(1986) Nature 21:522-525; Reichmann et al. (1988) Nature 332:323-329;EP-B-239 400 published 30 Sep. 1987; Presta (1992) Curr. Op. Struct.Biol. 2:593-596; and EP-B-451 216 published 24 Jan. 1996), whichreferences are herein incorporated by reference in their entirety. Thehumanized antibody includes a Primatized™ antibody wherein theantigen-binding region of the antibody is derived from an antibodyproduced by immunizing macaque monkeys with the antigen of interest.

By “neutralizing antibody” is meant an antibody molecule as hereindefined which is able to block or significantly reduce an effectorfunction of native sequence NRG3. For example, a neutralizing antibodymay inhibit or reduce the ability of NRG3 to activate an ErbB receptor,preferably an ErbB4 receptor, in the tyrosine phosphorylation assaydescribed herein. The neutralizing antibody may also block the mitogenicactivity of NRG3 in the cell proliferation assay disclosed herein.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567(Cabilly et al.; Morrison et al. (1984) Proc. Natl. Acad. Sci. USA81:6851-6855).

In the context of the present invention the expressions “cell”, “cellline”, and “cell culture” and “host cell” are used interchangeably, andall such designations include progeny. It is also understood that allprogeny may not be precisely identical in DNA content, due to deliberateor inadvertent mutations. Mutant progeny that have the same function orbiological property, as screened for in the originally transformed cell,are included. Methods of stable transfer, meaning that the foreign DNAis continuously maintained in the host, are known in the art.

The terms “replicable expression vector”, “expression vector” and“vector” refer to a piece of DNA, usually double-stranded, which mayhave inserted into it a piece of foreign DNA. Foreign DNA is defined asheterologous DNA, which is DNA not naturally found in the host cell. Thevector is used to transport the foreign or heterologous DNA into asuitable host cell. Once in the host cell, the vector can replicateindependently of the host chromosomal DNA, and several copies of thevector and its inserted (foreign) DNA may be generated. In addition, thevector contains the necessary elements that permit translating theforeign DNA into a polypeptide. Many molecules of the polypeptideencoded by the foreign DNA can thus be rapidly synthesized.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, a ribosomebinding site, and possibly, other as yet poorly understood sequences.Eukaryotic cells are known to utilize promoters, polyadenylationsignals, and enhancer.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or a secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used in accordwith conventional practice.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods,such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as those described in EP 266,032, published4 May 1988, or via deoxynucleoside H-phosphanate intermediates asdescribed by Froehler et al. (1986) Nucl. Acids Res. 14:5399. They arethen purified on polyacrylamide gels.

By “solid phase” is meant a non-aqueous matrix to which a reagent ofinterest (e.g., NRG3 or an antibody thereto) can adhere. Examples ofsolid phases encompassed herein include those formed partially orentirely of glass (e.g., controlled pore glass), polysaccharides (e.g.,agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.In certain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149, herein incorporated byreference in its entirety.

The terms “transformation” and “transfection” are used interchangeablyherein and refer to the process of introducing DNA into a cell.Following transformation or transfection, the NRG3 DNA may integrateinto the host cell genome, or may exist as an extrachromosomal element.If prokaryotic cells or cells that contain substantial cell wallconstructions are used as hosts, the preferred methods of transfectionof the cells with DNA is the calcium treatment method described by Cohenet al. (1972) Proc. Natl. Acad. Sci. U.S.A., 69:2110-2114 or thepolyethylene glycol method of Chung et al. (1988) Nuc. Acids. Res.16:3580. If yeast are used as the host, transfection is generallyaccomplished using polyethylene glycol, as taught by Hinnen (1978) Proc.Natl. Acad. Sci. U.S.A. 75:1929-1933. If mammalian cells are used ashost cells, transfection generally is carried out by the calciumphosphate precipitation method, Graham et al. (1978) Virology 52:546,Gorman et al. (1990) DNA and Protein Eng. Tech. 2:3-10. However, otherknown methods for introducing DNA into prokaryotic and eukaryotic cells,such as nuclear injection, electroporation, or protoplast fusion alsoare suitable for use in this invention.

Particularly useful in this invention are expression vectors thatprovide for the transient expression in mammalian cells of DNA encodingNRG3. In general, transient expression involves the use of an expressionvector that is able to efficiently replicate in a host cell, such thatthe host cell accumulates many copies of the expression vector and, inturn, synthesizes high levels of a desired polypeptide encoded by theexpression vector. Transient expression systems, comprising a suitableexpression vector and a host cell, allow for the convenient positiveidentification of polypeptides encoded by cloned DNAs, as well as forthe rapid screening of such polypeptides for desired biological orphysiological properties.

It is further envisioned that the NRG3 of this invention may be producedby homologous recombination, as provided for in WO 91/06667, published16 May 1991. Briefly, this method involves transforming a cellcontaining an endogenous NRG3 gene with a homologous DNA, whichhomologous DNA comprises (a) an amplifiable gene (e.g a gene encodingdihydrofolate reductase (DHFR)), and (b) at least one flanking sequence,having a length of at least about 150 base pairs, which is homologouswith a nucleotide sequence in the cell genome that is within or inproximity to the gene encoding NRG3. The transformation is carried outunder conditions such that the homologous DNA integrates into the cellgenome by recombination. Cells having integrated the homologous DNA arethen subjected to conditions which select for amplification of theamplifiable gene, whereby the NRG3 gene is amplified concomitantly. Theresulting cells are then screened for production of desired amounts ofNRG3. Flanking sequences that are in proximity to a gene encoding NRG3are readily identified, for example, by the method of genomic walking,using as a starting point the nucleotide sequence, or fragment thereof,of mouse NRG3 of FIG. 1 (SEQ ID NO:1), or human NRG3 of FIG. 2 (SEQ IDNO:5) or FIG. 3 (SEQ ID NO:22). DNA encoding the mouse and human NRG3polypeptides is deposited with the American Type Culture Collection asATCC 209156 (mouse; pLXSN.mNRG3), ATCC 209157 (human;pRK5.tk.neo.hNRG3B1), or ATCC 209297 (human; pRK5.tk.neo.hNRG3B2).

The expression “enhancing survival of a cell” refers to the act ofincreasing the period of existence of a cell, relative to an untreatedcell which has not been exposed to NRG3, either in vitro or in vivo.

The phrase “enhancing proliferation of a cell” encompasses the step ofincreasing the extent of growth and/or reproduction of the cell,relative to an untreated cell, either in vitro or in vivo. An increasein cell proliferation in cell culture can be detected by counting thenumber of cells before and after exposure to NRG3 (see the Examplebelow). The extent of proliferation can be quantified via microscopicexamination of the degree of confluency. Cell proliferation can also bequantified by measuring ³H uptake by the cells.

By “enhancing differentiation of a cell” is meant the act of increasingthe extent of the acquisition or possession of one or morecharacteristics or functions which differ from that of the original cell(i.e. cell specialization). This can be detected by screening for achange in the phenotype of the cell (e.g. identifying morphologicalchanges in the cell).

“Muscle cells” include skeletal, cardiac or smooth muscle tissue cells.This term encompasses those cells which differentiate to form morespecialized muscle cells (e.g. myoblasts).

“Isolated NRG3 nucleic acid” is RNA or DNA free from at least onecontaminating source nucleic acid with which it is normally associatedin the natural source and preferably substantially free of any othermammalian RNA or DNA. The phrase “free from at least one contaminatingsource nucleic acid with which it is normally associated” includes thecase where the nucleic acid is present in the source or natural cell butis in a different chromosomal location or is otherwise flanked bynucleic acid sequences not normally found in the source cell. An exampleof isolated NRG3 nucleic acid is RNA or DNA that encodes a biologicallyactive NRG3 sharing at least 75%, more preferably at least 80%, stillmore preferably at least 85%, even more preferably 90%, and mostpreferably 95% sequence identity with the mouse NRG3 shown in FIG. 1(SEQ ID NO:1), or human NRG3 shown in FIG. 2 (SEQ ID NO:4) or FIG. 3(SEQ ID NO:22).

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Hybridization is preferably performed under “stringent conditions” whichmeans (1) employing low ionic strength and high temperature for washing,for example, 0.015 sodium chloride/0.0015 M sodium citrate/0.1% sodiumdodecyl sulfate at 50° C., or (2) employing during hybridization adenaturing agent, such as formamide, for example, 50% (vol/vol)formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 nM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C. Another example is useof 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6/8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1%SDS. Yet another example is hybridization using a buffer of 10% dextransulfate, 2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C., followed by a high-stringency wash consisting of 0.1×SSC containingEDTA at 55° C.

“Immunoadhesins” or “NRG3—immunoglobulin chimeras” are chimericantibody-like molecules that combine the functional domain(s) of abinding protein (usually a receptor, a cell-adhesion molecule or aligand) with the an immunoglobulin sequence. The most common example ofthis type of fusion protein combines the hinge and Fc regions of animmunoglobulin (Ig) with domains of a cell-surface receptor thatrecognizes a specific ligand. This type of molecule is called an“immunoadhesin”, because it combines “immune” and “adhesion” functions;other frequently used names are “Ig-chimera”, “Ig-” or “Fc-fusionprotein”, or “receptor-globulin.”

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder of thosein which the disorder is to be prevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as sheep, dogs, horses, cats, cows, and the like.Preferably, the mammal herein is a human.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween™, polyethylene glycol (PEG), and Pluronics™.

General Procedures for the Production of an NRG3 by Recombinant DNATechnology

A. Identification and Isolation of Nucleic Acid Encoding NovelNeuregulin Related Ligand, NRG3.

The native NRG3s of the present invention may be isolated from cDNA orgenomic libraries. For example, a suitable cDNA library can beconstructed by obtaining polyadenylated mRNA from cells known to expressthe desired NRG3, and using the mRNA as a template to synthesize doublestranded cDNA. Suitable sources of the mRNA are embryonic and adultmammalian tissues. mRNA encoding native NRG3s of the present inventionis expressed, for example, in adult mammalian, brain, nervous system,heart, muscle, and testis. The gene encoding the novel NRG3s of thepresent invention can also be obtained from a genomic library, such as ahuman genomic cosmid library, or a mouse-derived embryonic stem cell(ES) genomic library.

Libraries, either cDNA or genomic, are screened with probes designed toidentify the gene of interest or the protein encoded by it. For cDNAexpression libraries, suitable probes include monoclonal and polyclonalantibodies that recognize and specifically bind to a NRG3 of theinvention. For cDNA libraries, suitable probes include carefullyselected oligonucleotide probes (usually of about 20-80 bases in length)that encode known or suspected portions of a NRG3 polypeptide from thesame or different species, and/or complementary or homologous cDNAs orfragments thereof that encode the same or a similar gene. Appropriateprobes for screening genomic DNA libraries include, without limitation,oligonucleotides, cDNAs, or fragments thereof that encode the same or asimilar gene, and/or homologous genomic DNAs or fragments thereof.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures as described in Chapters 10-12 ofSambrook et al., Molecular Cloning: A Laboratory Manual, New York, ColdSpring Harbor Laboratory Press, 1989, herein incorporated by referencein its entirety.

If DNA encoding a NRG3 of the present invention is isolated by usingcarefully selected oligonucleotide sequences to screen cDNA librariesfrom various tissues, the oligonucleotide sequences selected as probesshould be sufficient in length and sufficiently unambiguous that falsepositive selections are minimized. The actual nucleotide sequence(s)is/are usually designed based on regions that have the least codonredundance. The oligonucleotides may be degenerate at one or morepositions. The use of degenerate oligonucleotides is of particularimportance where a library is screened from a species in whichpreferential codon usage is not known.

The oligonucleotide must be labeled such that it can be detected uponhybridization to DNA in the library being screened. The preferred methodof labeling is to use ATP (e.g., γ³²P) and polynucleotide kinase toradiolabel the 5′ end of the oligonucleotide. However, other methods maybe used to label the oligonucleotide, including, but not limited to,biotinylation or enzyme labeling.

cDNAs encoding the novel NRG3s can also be identified and isolated byother known techniques of recombinant DNA technology, such as by directexpression cloning, or by using the polymerase chain reaction (PCR) asdescribed in U.S. Pat. No. 4,683,195, issued 28 Jul. 1987, in section 14of Sambrook et al., supra, or in Chapter 15 of Current Protocols inMolecular Biology, Ausubel et al. eds., Greene Publishing Associates andWiley-Interscience 1991, which references are herein incorporated byreference in their entirety.

Once cDNA encoding a new native ErbB4 receptor-specific NRG3 from onespecies has been isolated, cDNAs from other species can also be obtainedby cross-species hybridization. According to this approach, human orother mammalian cDNA or genomic libraries are probed by labeledoligonucleotide sequences selected from known NRG3 sequences (such asmurine or human sequences) in accord with known criteria. Preferably,the probe sequence should be sufficient in length and sufficientlyunambiguous that false positives are minimized. Typically, a ³²P-labeledoligonucleotide having about 30 to 50 bases is sufficient, particularlyif the oligonucleotide contains one or more codons for methionine ortryptophan. Isolated nucleic acid will be DNA that is identified andseparated from contaminant nucleic acid encoding other polypeptides fromthe source of nucleic acid. Hybridization is preferably performed under“stringent conditions”, as defined herein.

Once the sequence is known, the gene encoding a particular NRG3 can alsobe obtained by chemical synthesis, following one of the methodsdescribed in Engels and Uhlmann, Agnew (1989) Chem. Int. Ed. Engl.28:716, herein incorporated by reference in its entirety. These methodsinclude triester, phosphite, phosphoramidite and H-phosphonate methods,PCR and other autoprimer methods, and oligonucleotide syntheses on solidsupports.

B. Cloning and Expression of Nucleic Acid Encoding the Novel NRG3s.

Once the nucleic acid encoding a novel NRG3 is available, it isgenerally ligated into a replicable expression vector for furthercloning (amplification of the DNA), or for expression.

Expression and cloning vectors are well known in the art and contain anucleic acid sequence that enables the vector to replicate in one ormore selected host cells. The selection of the appropriate vector willdepend on 1) whether it is to be used for DNA amplification or for DNAexpression, 2) the size of the DNA to be inserted into the vector, and3) the host cell to be transformed with the vector. Each vector containsvarious components depending on its function (amplification of DNA ofexpression of DNA) and the host cell for which it is compatible. Thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of the above listed components, the desired coding and controlsequences, employs standard ligation techniques. Isolated plasmids orDNA fragments are cleaved, tailored, and religated in the form desiredto generate the plasmids required. For analysis to confirm correctsequences in plasmids constructed, the ligation mixtures are commonlyused to transform E. coli cells, e.g. E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al. (1981)Nucleic Acids Res. 9:309 or by the method of Maxam et al. (1980) Methodsin Enzymology 65:499.

The polypeptides of the present invention may be expressed in a varietyof prokaryotic and eukaryotic host cells. Suitable prokaryotes includegram negative or gram positive organisms, for example E. coli orbacilli. A preferred cloning host is E. coli 294 (ATCC 31,446) althoughother gram negative or gram positive prokaryotes such as E. coil B, E.coli X1776 (ATCC 31,537), E. coli W3110 (ATCC 27,325), Pseudomonasspecies, or Serratia Marcesans are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for vectors herein. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms. However, a number of other genera,species and strains are commonly available and useful herein, such as S.pombe (Beach and Nurse (1981) Nature 290:140), Kluyveromyces lactis(Louvencourt et al. (1983) J. Bacteriol. 737); yarrowla (EP 402,226);Pichia pastoris (EP 183,070), Trichoderma reesia (EP 244,234),Neurospora crassa (Case et al. (1979) Proc. Natl. Acad. Sci. USA76:5259-5263); and Aspergillus hosts such as A. nidulans (Ballance etal. (1983) Biochem. Biophys. Res. Commun. 112:284-289; Tilburn et al.(1983) Gene 26:205-221; Yelton et al. (1984) Proc. Natl. Acad. Sci. USA81:1470-1474) and A. niger (Kelly and Hynes (1985) EMBO J. 4:475479).

Suitable host cells may also derive from multicellular organisms. Suchhost cells are capable of complex processing and glycosylationactivities. In principle, any higher eukaryotic cell culture isworkable, whether from vertebrate or invertebrate culture, althoughcells from mammals such as humans are preferred. Examples ofinvertebrate cells include plants and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melangaster(fruitfly), and Bombyx mori host cells have been identified. See, e.g.Luckow et al. (1988) Bio/Technology 6:47-55; Miller et al., in GeneticEngineering, Setlow, J. K. et al., eds., Vol. 8 (Plenum Publishing,1986), pp. 277-279; and Maeda et al. (1985) Nature 315:592-594. Avariety of such viral strains are publicly available, e.g. the L-1variant of Autographa californica NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the NRG3 DNA. During incubation of the plant cell culture withA. tumefaciens, the DNA encoding a NRG3 is transferred to the plant cellhost such that it is transfected, and will, under appropriateconditions, express the NRG3 DNA. In addition, regulatory and signalsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.Depicker et al. (1982) J. Mol. Appl. Gen. 1:561. In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. SeeEP 321,196 published 21 Jun. 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) is per se well known(see for example, Tissue Culture, Academic Press, Kruse and Patterson,editors (1973)). Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney cell line (293 or 293 cells subcloned for growth insuspension culture, Graham et al. (1977) J. Gen. Virol. 36:59); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA77:4216); mouse sertolli cells (TM4, Mather (1980) Biol. Reprod.23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCCCCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al. (1982) Annals N.Y. Acad.Sci. 383:44068); MRC 5 cells; FS4 cells; and a human hepatoma cell line(Hep G2). Preferred host cells are human embryonic kidney 293 andChinese hamster ovary cells.

Particularly useful in the practice of this invention are expressionvectors that provide for the expression in mammalian cells of DNAencoding a novel NRG3 herein. Where transient expression is preferred,expression involves the use of an expression vector that is able toreplicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector. Transient systems, comprising a suitable expressionvector and a host cell, allow for the convenient positive identificationof polypeptides encoded by cloned DNAs, as well as for the rapidscreening of such polypeptides for desired biological or physiologicalproperties. Thus, transient expression systems are particularly usefulin the invention for purposes of identifying analogs and variants of anative NRG3 of the invention.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the NRG3s in recombinant vertebrate cell culture aredescribed for example, in Getting et al. (1981) Nature 293:620-625;Mantel et al. (1979) Nature 281:4046; Levinson et al.; EP 117,060 and EP117,058. Particularly useful plasmids for mammalian cell cultureexpression of the NRG3 polypeptides are pRK5 (EP 307,247), or pSVI6B(PCT Publication No. WO 91/08291).

Other cloning and expression vectors suitable for the expression of theNRG3s of the present invention in a variety of host cells are, forexample, described in EP 457,758 published 27 Nov. 1991. A large varietyof expression vectors is now commercially available. An exemplarycommercial yeast expression vector is pPIC.9 (Invitrogen), while ancommercially available expression vector suitable for transformation ofE. coli cells is PET15b (Novagen).

C. Culturing the Host Cells.

Prokaryote cells used to produced the NRG3s of this invention arecultured in suitable media as describe generally in Sambrook et al.,supra.

Mammalian cells can be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium(MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium(DMEM, Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace (1979) Meth. Enzymol.58:44; Barnes and Sato (1980) Anal. Biochem. 102:255, U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195or US Pat. Re. 30,985 may be used as culture media for the host cells.Any of these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug) trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH and the like, suitably arethose previously used with the host cell selected for cloning orexpression, as the case may be, and will be apparent to the ordinaryartisan.

The host cells referred to in this disclosure encompass cells in invitro cell culture as well as cells that are within a host animal orplant.

It is further envisioned that the NRG3s of this invention may beproduced by homologous recombination, or with recombinant productionmethods utilizing control elements introduced into cells alreadycontaining DNA encoding the particular NRG3.

D. Detecting Gene Amplification and/or Expression.

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas (1980) Proc.Natl. Acad. Sci. USA 77:5201-5205), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as a site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to thesurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. A particularly sensitive staining techniquesuitable for use in the present invention is described by Hse et al.(1980) Am. J. Clin. Pharm. 75:734-738.

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any animal. Conveniently, the antibodies may be preparedagainst a native NRG3 polypeptide, or against a synthetic peptide basedon the DNA sequence disclosed herein.

E. Amino Acid Sequence Variants of a Native NRG3.

Amino acid sequence variants of native NRG3s are prepared by methodsknown in the art by introducing appropriate nucleotide changes into anative NRG3 DNA, or by in vitro synthesis of the desired polypeptide.There are two principal variables in the construction of amino acidsequence variants: the location of the mutation site and the nature ofthe mutation. With the exception of naturally-occurring alleles, whichdo not require the manipulation of the DNA sequence encoding the nativeNRG3, the amino acid sequence variants of NRG3s are preferablyconstructed by mutating the DNA, either to arrive at an allele or anamino acid sequence variant that does not occur in nature.

One group of mutations will be created within the extracellular domainor within the EGF-like domain of a novel native mouse or human NRG3 ofthe present invention (see FIG. 3 for the delineation of theextracellular domain (SEQ ID NO:3 or SEQ ID NO:7) and EGF-like domain(SEQ ID NO:4) within human or mouse NRG3 amino acid sequences,respectively. Since these domains are believed to be functionallyimportant, alterations such as non-conservative substitutions,insertions and/or deletions in these regions are expected to result ingenuine changes in the properties of the native receptor molecules suchas in ErbB4 receptor binding and activation. Accordingly, amino acidalterations in this region are also believed to result in variants withproperties significantly different from the corresponding nativepolypeptides. Non-conservative substitutions within these functionallyimportant domains may result in variants which lose the ErbB4 receptorrecognition and binding ability of their native counterparts, or haveincreased ErbB4 receptor recognition properties, enhanced selectivity,or enhanced activation properties as compared to the correspondingnative proteins.

Alternatively or in addition, amino acid alterations can be made atsites that differ in novel NRG3s from various species, or in highlyconserved regions, depending on the goal to be achieved. Sites at suchlocations will typically be modified in series, e.g. by (1) substitutingfirst with conservative choices and then with more radical selectionsdepending upon the results achieved, (2) deleting the target residue orresidues, or (3) inserting residues of the same or different classadjacent to the located site, or combinations of options 1-3. Onehelpful technique for such modifications is called “alanine scanning”(Cunningham and Wells (1989) Science 244:1081-1085).

In yet another group of the variant NRG3s of the present invention, oneor more of the functionally less significant domains may be deleted orinactivated. For example, the deletion or inactivation of thetransmembrane domain yields soluble variants of the native proteins.Alternatively, or in addition, the cytoplasmic domain may be deleted,truncated or otherwise altered.

Naturally-occurring amino acids are divided into groups based on commonside chain properties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophobic: cys, ser, thr;    -   (3) acidic: asp, glu;    -   (4) basic: asn, gln, his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Conservative substitutions involve exchanging a member within one groupfor another member within the same group, whereas non-conservativesubstitutions will entail exchanging a member of one of these classesfor another. Substantial changes in function or immunological identityare made by NRG3 substitutions that are less conservative, i.e. differmore significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of substitution, for example as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site or (c) the bulk of the side chain. Thesubstitutions which in general are expected to produce the greatestchanges in the properties of the novel native NRG3s of the presentinvention will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine. Such substitutions are expected to have their mostsignificant effect when made within the extracellular domain, such as inthe EGF-like domain.

Substitutional variants of the novel NRG3s of the present invention alsoinclude variants where functionally homologous (having at least about40%-50% homology) domains of other proteins are substituted by routinemethods for one or more of the above-identified domains within the novelNRG3 structure, such as the extracellular domain or EGF-like domain.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Typically, the transmembrane and cytoplasmic domains, oronly the transmembrane domains are deleted. However, deletion from theC-terminus to any suitable amino acid N-terminal to the transmembraneregion which preserves the biological activity or immunologicalcross-reactivity of a native NRG3 is suitable. The transmembrane region(TM) of each of the human and mouse NRG3 consensus sequences is shown inFIGS. 4A and 4B to range from about amino acid 362 to about amino acid384 (human SEQ ID NO:6 and SEQ ID NO:23), and about amino acid 360 toabout amino acid 382 (mouse SEQ ID NO:2).

A preferred class of substitutional and/or deletional variants of thepresent invention are those involving a transmembrane region of a novelNRG3 molecule. Transmembrane regions are highly hydrophobic orlipophilic domains that are the proper size to span the lipid bilayer ofthe cellular membrane. They are believed to anchor the NRG3 in the cellmembrane, and allow for homo- or heteropolymeric complex formation.Inactivation of the transmembrane domain, typically by deletion orsubstitution of transmembrane domain hydroxylation residues, willfacilitate recovery and formulation by reducing its cellular or membranelipid affinity and improving its aqueous solubility. If thetransmembrane and cytoplasmic domains are deleted one avoids theintroduction of potentially immunogenic epitopes, whether by exposure ofotherwise intracellular polypeptides that might be recognized by thebody as foreign or by insertion of heterologous polypeptides that arepotentially immunogenic. Inactivation of the membrane insertion functionis accomplished by deletion of sufficient residues to produce asubstantially hydrophilic hydropathy profile in the transmembrane or bysubstituting with heterologous residues which accomplish the sameresult.

A principle advantage of the transmembrane inactivated variants of theNRG3s of the present invention is that they may be secreted into theculture medium of recombinant hosts. These variants are soluble in bodyfluids such as blood and do not have an appreciable affinity for cellmembrane lipids, thus considerably simplifying their recovery fromrecombinant cell culture. As a general proposition, such solublevariants will retain a functional extracellular domain or fragmentthereof, will not have a functional transmembrane domain, and preferablywill not have a functional cytoplasmic domain.

For example, the transmembrane domain may be substituted by any aminoacid sequence, e.g. a random or predetermined sequences of about 5 to 50serine, threonine, lysine, arginine, glutamine, aspartic acid and likehydrophilic residues, which altogether exhibit a hydrophilic hydropathyprofile. Like the deletional (truncated) soluble variants, thesevariants are secreted into the culture medium of recombinant hosts.

Amino acid insertions include amino- and/or carboxyl-terminal fusionsranging in length from one residue to polypeptides containing a hundredor more residues, as well as intrasequence insertions of single ormultiple amino acid residues. Intrasequence insertions (i.e. insertionswithin the novel NRG3 amino acid sequence) may range generally fromabout 1 to 10 residues, more preferably 1 to 5 residues, more preferably1 to 3 residues. An example of a terminal insertion includes fusion of aheterologous N-terminal signal sequence to the N-terminus of the NRG3molecule to facilitate the secretion of the mature NRG3 or a fragmentthereof from recombinant host cells. Such signal sequences willgenerally be obtained from, and thus be homologous to, a signal sequenceof the intended host cell species. Suitable sequences include STII orIpp for E. coli, alpha factor for yeast, and viral signals such asherpes gD for mammalian cells.

Other insertional variants of the native NRG3 molecules include thefusion of the N- or C-terminus of the NRG3 molecule to immunogenicpolypeptides, e.g. bacterial polypeptides such as beta-lactamase or anenzyme encoded by the E. coli trp locus, or yeast protein, andC-terminal fusions with proteins having a long half-life such asimmunoglobulin regions (preferably immunoglobulin constant regions),albumin, or ferritin, as described in WO 89/02922 published on 6 Apr.1989.

Further insertional variants are immunologically active derivatives ofthe novel NRG3s, which comprise the EGF-like domain and a polypeptidecontaining an epitope of an immunologically competent extraneouspolypeptide, i.e. a polypeptide which is capable of eliciting an immuneresponse in the animal to which the fusion is to be administered orwhich is capable of being bound by an antibody raised against anextraneous polypeptide. Typical examples of such immunologicallycompetent polypeptides are allergens, autoimmune epitopes, or otherpotent immunogens or antigens recognized by pre-existing antibodies inthe fusion recipient, including bacterial polypeptides such as trpLE,β-glactosidase, viral polypeptides such as herpes gD protein, and thelike.

Immunogenic fusions are produced by cross-linking in vitro or by cultureof cells transformed with recombinant DNA encoding an immunogenicpolypeptide. It is preferable that the immunogenic fusion be one inwhich the immunogenic sequence is joined to or inserted into a novelNRG3 molecule or fragment thereof by one or more peptide bonds. Theseproducts therefore consist of a linear polypeptide chain containing theNRG3 epitope and at least one epitope foreign to the NRG3. It will beunderstood that it is within the scope of this invention to introducethe epitopes anywhere within a NRG3 molecule of the present invention ora fragment thereof. These immunogenic insertions are particularly usefulwhen formulated into a pharmacologically acceptable carrier andadministered to a subject in order to raise antibodies against the NRG3molecule, which antibodies in turn are useful as diagnostics, intissue-typing, or in purification of the novel NRG3s by standardimmunoaffinity techniques. Alternatively, in the purification of theNRG3s of the present invention, binding partners for the fusedextraneous polypeptide, e.g. antibodies, receptors or ligands, are usedto adsorb the fusion from impure admixtures, after which the fusion iseluted and, if desired, the novel NRG3 is recovered from the fusion,e.g. by enzymatic cleavage.

Since it is often difficult to predict in advance the characteristics ofa variant NRG3, it will be appreciated that some screening will beneeded to select the optimum variant.. Such screening includes, but isnot limited to, arrays of ErbB4 receptor binding.

After identifying the desired mutation(s), the gene encoding a NRG3variant can, for example, be obtained by chemical synthesis as describedherein. More preferably, DNA encoding a NRG3 amino acid sequence variantis prepared by site-directed mutagenesis of DNA that encodes an earlierprepared variant or a nonvariant version of the NRG3. Site-directed(site-specific) mutagenesis allows the production of NRG3 variantsthrough the use of specific oligonucleotide sequences that encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 20 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered. In general, thetechniques of site-specific mutagenesis are well known in the art, asexemplified by publications such as, Edelman et al. (1983) DNA 2:183. Aswill be appreciated, the site-specific mutagenesis technique typicallyemploys a phage vector that exists in both a single-stranded anddouble-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam (1981). Thisand other phage vectors are commercially available and their use is wellknown to those skilled in the art. A versatile and efficient procedurefor the construction of oligodeoxyribonucleotide directed site-specificmutations in DNA fragments using M13-derived vectors was published byZoller, M. J. and Smith, M. (1982) Nucleic Acids Res. 10:6487-6500).Also, plasmid vectors that contain a single-stranded phage origin ofreplication (Veira et al. (1987) Meth. Enzymol. 153:3) may be employedto obtain single-stranded DNA. Alternatively, nucleotide substitutionsare introduced by synthesizing the appropriate DNA fragment in vitro,and amplifying it by PCR procedures known in the art.

The PCR amplification technique may also be used to create amino acidsequence variants of a novel NRG3. In a specific example of PCRmutagenesis, template plasmid DNA (1 μg) is linearized by digestion witha restriction endonuclease that has a unique recognition site in theplasmid DNA outside of the region to be amplified. Of this material, 100ng is added to a PCR mixture containing PCR buffer, which contains thefour deoxynucleotide triphosphates and is included in the GeneAmp^(R)kits (obtained from Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville,Calif.), and 25 pmole of each oligonucleotide primer, to a final volumeof 50 μl. The reaction mixture is overlayered with 35 μl mineral oil.The reaction is denatured for 5 minutes at 100° C., placed briefly onice, and then 1 μl Thermus aquaticus (Taq) DNA polymerase (5 units/1),purchased from Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville,Calif.) is added below the mineral oil layer. The reaction mixture isthen inserted into a DNA Thermal Cycler (Perkin-Elmer Cetus) programmedas follows: (as an example)

-   -   2 min. 55° C.,    -   30 sec. 72° C., then 19 cycles of the following:    -   30 sec. 94° C.,    -   30 sec. 55° C., and    -   30 sec. 72° C.

At the end of the program, the reaction vial is removed from the thermalcycler and the aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to appropriate treatments for insertion into a vector.

Cassette mutagenesis is another method useful for preparing variants andis based on the technique described by Wells et al. (1985) Gene 34:315.

Additionally, the so-called phagemid display method may be useful inmaking amino acid sequence variants of native or variant NRG3s or theirfragments. This method involves 1) constructing a replicable expressionvector comprising a first gene encoding a receptor to be mutated, asecond gene encoding at least a portion of a natural or wild-type phagecoat protein wherein the first and second genes are heterologous, and atranscription regulatory element operably linked to the first and secondgenes, thereby forming a gene fusion encoding a fusion protein; 2)mutating the vector at one or more selected positions within the firstgene thereby forming a family of related plasmids; 3) transformingsuitable host cells with the plasmids; 4) infecting the transformed hostcells with a helper phage having a gene encoding the phage coat protein;5) culturing the transformed infected host cells under conditionssuitable for forming recombinant phagemid particles containing at leasta portion of the plasmid and capable of transforming the host, theconditions adjusted so that no more than a minor amount of phagemidparticles display more than one copy of the fusion protein on thesurface of the particle; 6) contacting the phagemid particles with asuitable antigen so that at least a portion of the phagemid particlesbind to the antigen; and 7) separating the phagemid particles that bindfrom those that do not. Steps 4 through 7 can be repeated one or moretimes. Preferably in this method the plasmid is under tight control ofthe transcription regulatory element, and the culturing conditions areadjusted so that the amount or number of phagemid particles displayingmore than one copy of the fusion protein on the surface of the particleis less than about 1%. Also, preferably, the amount of phagemidparticles displaying more than one copy of the fusion protein is lessthan 10% of the amount of phagemid particles displaying a single copy ofthe fusion protein. Most preferably, the amount is less than 20%.Typically in this method, the expression vector will further contain asecretory signal sequence fused to the DNA encoding each subunit of thepolypeptide and the transcription regulatory element will be a promotersystem. Preferred promoter systems are selected from Zac Z, λ_(PL), tac,T7 polymerase, tryptophan, and alkaline phosphatase promoters andcombinations thereof. Also, normally the method will employ a helperphage selected from M13K07, M13R408, M13-VCS, and Phi X 174. Thepreferred helper phage is M13K07, and the preferred coat protein is theMl3 3Phage gene III coat protein. The preferred host is E. coli, andprotease-deficient strains of E. coli.

Further details of the foregoing and similar mutagenesis techniques arefound in general textbooks, such as, for example, Sambrook et al.,supra, and Current Protocols in Molecular Biology, Ausubel et al. eds.,supra.

F. Glycosylation Variants.

Glycosylation variants are included within the scope of the presentinvention. They include variants completely lacking in glycosylation(unglycosylated), variants having at least one less glycosylated sitethan the native form (deglycosylated) as well as variants in which thegycosylation has been changed. Included are deglycosylated andunglycosylated amino acid sequences variants, deglycosylated andunglycosylated native NRG3s or fragments thereof and other glycosylationvariants. For example, substitutional or deletional mutagenesis may beemployed to eliminate the N- or O-linked glycosylation sites in the anative or variant NRG3 of the present invention, e.g. the asparagineresidue may be deleted or substituted for another basic residue such aslysine or histidine. Alternatively, flanking residues making up theglycosylation site may be substituted or deleted, even though theasparagine residues remain unchanged, in order to prevent glycosylationby eliminating the glycosylation recognition site. Where the preferredNRL variant is the EGF-like domain of NRG3, the fragment is preferablyunglycosylated.

Additionally, unglycosylated NRG3s which have the glycosylation sites ofa native molecule may be produced in recombinant prokaryotic cellculture because prokaryotes are incapable of introducing glycosylationinto polypeptides.

Glycosylation variants may be produced by appropriate host cells or byin vitro methods. Yeast and insect cells, for example, introduceglycosylation which varies significantly from that of mammalian systems.Similarly, mammalian cells having a different species (e.g. hamster,murine, porcine, bovine or ovine), or tissue origin (e.g. lung, liver,lymphoid, mesenchymal or epidermal) than the source of the NRG3 areroutinely screened for the ability to introduce variant glycosylation ascharacterized for example by elevated levels of mannose or variantratios of mannose, fucose, sialic acid, and other sugars typically foundin mammalian glycoproteins. In vitro processing of the NRG3 typically isaccomplished by enzymatic hydrolysis, e.g. neuraminidate digestion.

G. Covalent Modifications.

Covalent modifications of the novel NRG3s of the present invention areincluded within the scope of the invention. Such modifications aretraditionally introduced by reacting targeted amino acid residues of theNRG3s with an organic derivatizing agent that is capable of reactingwith selected amino acid side chains or terminal residues, or byharnessing mechanisms of post-translational modifications that functionin selected recombinant host cells. The resultant covalent derivativesare useful in programs directed at identifying residues important forbiological activity, for immunoassays of the NRG3, or for thepreparation of anti-NRG3 antibodies for immunoaffinity purification ofthe recombinant. For example, complete inactivation of the biologicalactivity of the protein after reaction with ninhydrin would suggest thatat least one arginyl or lysyl residue is critical for its activity,whereafter the individual residues which were modified under theconditions selected are identified by isolation of a peptide fragmentcontaining the modified amino acid residue. Such modifications arewithin the ordinary skill in the art and are performed without undueexperimentation.

Derivatization with bifunctional agents is useful for preparingintramolecular aggregates of the NRG3s with polypeptides as well as forcross-linking the NRG3 polypeptide to a water insoluble support matrixor surface for use in assays or affinity purification. In addition, astudy of interchain cross-links will provide direct information onconformational structure. Commonly used cross-linking agents include1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, homobifunctional imidoesters, andbifunctional maleimides. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates which are capable of forming cross-links in the presenceof light. Alternatively, reactive water insoluble matrices such ascyanogen bromide activated carbohydrates and the systems reactivesubstrates described in U.S. Pat. Nos. 3,959,642; 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employedfor protein immobilization and cross-linking.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andaspariginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl, threonyl ortyrosyl residues, methylation of the α-amino groups of lysine, arginine,and histidine side chains (T. E. Creighton (1983) Proteins: Structureand Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86).

Further derivatives of the NRG3s herein are the so called“immunoadhesins”, which are chimeric antibody-like molecules combiningthe functional domain(s) of a binding protein (usually a receptor, acell-adhesion molecule or a ligand) with the an immunoglobulin sequence.The most common example of this type of fusion protein combines thehinge and Fc regions of an immunoglobulin (1 g) with domains of acell-surface receptor that recognizes a specific ligand. This type ofmolecule is called an “immunoadhesin”, because it combines “immune” and“adhesion” functions; other frequently used names are “Ig-chimera”,“Ig-” or “Fc-fusion protein”, or “receptor-globulin.”

Immunoadhesins reported in the literature include, for example, fusionsof the T cell receptor (Gascoigne et al. (1987) Proc. Natl. Acad. Sci.USA 84:2936-2940); CD4 (Capon et al. (1989) Nature 337:525-531;Traunecker et al. (1989) Nature 3:68-70; Zettmeissl et al. (1990) DNACell Biol. USA 9:347-353; Byrn et al. (1990) Nature 344:667-670);L-seNRG3 (homing receptor) (Watson et al. (1990) J. Cell. Biol.110:2221-2229); Watson et al. (1991) Nature 349:164-167); E-seNRG3(Mulligan et al. (1993) J. Immunol. 151:6410-17; Jacob et al. (1995)Biochemistry 34:1210-1217); P-seNRG3 (Mulligan et al., supra;Hollenbaugh et al. (1995) Biochemistry 34:5678-84); ICAM-1 (Stauton etal. (1992) J. Exp. Med. 176:1471-1476; Martin et al. (1993) J. Virol.67:3561-68; Roep et al. (1994) Lancet 343:1590-93); ICAM-2 (Damle et al.(1992) J. Immunol. 148:665-71); ICAM-3 (Holness et al. (1995) J. Biol.Chem. 270:877-84); LFA-3 (Kanner et al. (1992) J. Immunol. 148:23-29);L1 glycoprotein (Doherty et al. (1995) Neuron 14:57-66); TNF-R1(Ashkenazi et al., (1991) Proc. Natl. Acad. Sci. USA 88:10535-539);Lesslauer et al. (1991) Eur. J. Immunol. 21:2883-86; Peppel et al (1991)J. Exp. Med. 174:1483-1489); TNF-R2 (Zack et al. (1993) Proc. Natl.Acad. Sci. USA 90:2335-39; Wooley et al. (1993) J. Immunol.151:6602-07); CD44 (Aruffo et al. (1990) Cell 61:1303-1313); CD28 and B7(Linsley et al. (1991) J. Exp. Med. 173:721-730); CTLA-4 (Lisley et al(1991) J. Exp. Med. 174:561-569); CD22 (Stamenkovic et al. (1991) Cell66:1133-1144); NP receptors (Bennett et al. (1991) J. Biol. Chem.266:23060-23067); IgE receptor α (Ridgway and Gorman (1991) J. Cell.Biol. 115:1448 abstr.); IFN-γR α- and β-chain (Marsters et al. (1995)Proc. Natl. Acad. Sci. USA 92:5401-05); trk-A, -B, and -C (Shelton etal. (1995) J. Neurosci. 15:477-91); IL-2 (Landolfi (1991) J. Immunol.146:915-19); IL-10 (Zheng et al. (1995) J. Immunol. 154:5590-5600).

The simplest and most straightforward immunoadhesin design combines thebinding region(s) of the ‘adhesin’ protein with the hinge and Fc regionsof an immunoglobulin heavy chain. Ordinarily, when preparing theNRG3-immunoglobulin chimeras of the present invention, nucleic acidencoding the desired NRG3 polypeptide will be fused at the C-terminus ofthe desired sequence to the N-terminus of a nucleic acid sequenceencoding an immunoglobulin constant domain sequence, however fusion tothe N-terminus of the desired NRG3 sequence is also possible. Typically,in such fusions the encoded chimeric polypeptide will retain at leastfunctionally active hinge, CH2 and CH3 domains of the constant region ofan immunoglobulin heavy chain. Fusions are also made to the C-terminusof the Fc portion of a constant domain, or immediately N-terminal to theCH1 of the heavy chain or the corresponding region of the light chain.The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize thebiological activity, secretion or binding characteristics of theNRG3-immunoglobulin chimeras.

In a preferred embodiment, the sequence of a native, mature NRG3polypeptide, or a soluble form thereof such as a (transmembranedomain-inactivated or EGF-like domain polypeptide) form thereof, isfused to the N-terminus of the C-terminal portion of an antibody (inparticular the Fc domain), containing the effector functions of animmunoglobulin, e.g. IgG-1. It is possible to fuse the entire heavychain constant region to the NRG3 sequence. However, more preferably, asequence beginning in the hinge region just upstream of the papaincleavage site (which defines IgG Fc chemically; residue 216, taking thefirst residue of heavy chain constant region to be 114 (Kobet et al.,supra), or analogous sites of other immunoglobulins) is used in thefusion. In a particularly preferred embodiment, the NRG3 sequence (fulllength or soluble) is fused to the hinge region and CH2 and CH3 or CH1,hinge, CH2 and CH3 domains of an IgG-1, IgG-2, or IgG-3 heavy chain. Theprecise site at which the fusion is made is not critical, and theoptimal site can be determined by routine experimentation.

In some embodiments, the NRG3-immunoglobulin chimeras are assembled asmultimers, and particularly as homo-dimers or -tetramers (WO 91/08298).Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four unit is repeated in the higher molecularweight immunoglobulins; IgM generally exists as a pentamer of basic fourunits held together by disulfide bonds. IgA globulin, and occasionallyIgG globulin, may also exist in multimeric form in serum. In the case ofmultimer, each four unit may be the same or different.

Various exemplary assembled NRG3-immunoglobulin chimeras within thescope of the invention are schematically diagrammed below:

-   -   (a) AC_(L)-AC_(L);    -   (b) AC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), or        V_(L)C_(L)-AC_(H)];    -   (c) AC_(L)-AC_(H)-[AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C^(H)];    -   (d) AC_(L)-V_(H)C_(H)-[AC_(H), or AC_(L)-V_(H)C_(H), or        V_(L)C_(L)-AC_(H)];    -   (e) V_(L)C_(L)-AC_(H)-[AC_(L)-V_(H)C_(H), or V_(L)C_(L)-AC_(H)];        and    -   (f) [A-Y]_(n)-[V_(L)C_(L)-V_(H)C_(H)]₂,        wherein    -   each A represents identical or different novel NRG3 polypeptide        amino acid sequences;    -   V_(L) is an immunoglobulin light chain variable domain;    -   V_(H) is an immunoglobulin heavy chain variable domain;    -   C_(L) is an immunoglobulin light chain constant domain;    -   C_(H) is an immunoglobulin heavy chain constant domain;    -   n is an integer greater than 1;    -   Y designates the residue of a covalent cross-linking agent.

In the interest of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed asbeing present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to anNRG3-immunoglobulin heavy chain fusion polypeptide, or directly fused tothe NRG3 polypeptide. In the former case, DNA encoding an immunoglobulinlight chain is typically coexpressed with the DNA encoding theNRG3-immunoglobulin heavy chain fusion protein. Upon secretion, thehybrid heavy chain and the light chain will be covalently associated toprovide an immunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs. Methods suitable for thepreparation of such structures are, for example, disclosed in U.S. Pat.No. 4,816,567 issued 28 Mar. 1989.

In a preferred embodiment, the immunoglobulin sequences used in theconstruction of the immunoadhesins of the present invention are from anIgG immunoglobulin heavy chain constant domain. For humanimmunoadhesins, the use of human IgG-1 and IgG-3 immunoglobulinsequences is preferred. A major advantage of using IgG-1 is that IgG-1immunoadhesins can be purified efficiently on immobilized protein A. Incontrast, purification of IgG-3 requires protein G, a significantly lessversatile medium. However, other structural and functional properties ofimmunoglobulins should be considered when choosing the Ig fusion partnerfor a particular immunoadhesin construction. For example, the IgG-3hinge is longer and more flexible, so it can accommodate larger‘adhesin’ domains that may not fold or function properly when fused toIgG-1. While IgG immunoadhesins are typically mono- or bivalent, otherIg subtypes like IgA and IgM may give rise to dimeric or pentamericstructures, respectively, of the basic Ig homodimer unit. Multimericimmunoadhesins are advantageous in that they can bind their respectivetargets with greater avidity than their IgG-based counterparts. Reportedexamples of such structures are CD4-IgM (Traunecker et al., supra);ICAM-IgM (Martin et al. (1993) J. Virol. 67:3561-68); and CD2-IgM(Arulanandam et al. (1993) J. Exp. Med. 177:1439-50).

For NRG3-Ig immunoadhesins, which are designed for in vivo application,the pharmacokinetic properties and the effector functions specified bythe Fc region are important as well. Although IgG-1, IgG-2 and IgG-4 allhave in vivo half-lives of 21 days, their relative potencies atactivating the complement system are different. IgG4 does not activatecomplement, and IgG-2 is significantly weaker at complement activationthan IgG-1. Moreover, unlike IgG-1, IgG-2 does not bind to Fc receptorson mononuclear cells or neutrophils. While IgG-3 is optimal forcomplement activation, its in vivo half-life is approximately one thirdof the other IgG isotypes. Another important consideration forimmunoadhesins designed to be used as human therapeutics is the numberof allotypic variants of the particular isotype. In general, IgGisotypes with fewer serologically-defined allotypes are preferred. Forexample, IgG-1 has only four serologically-defined allotypic sites, twoof which (G1m and 2) are located in the Fc region; and one of thesesites G1m1, is non-immunogenic. In contrast, there are 12serologically-defined allotypes in IgG-3, all of which are in the Fcregion; only three of these sites (G3m5, 11 and 21) have one allotypewhich is nonimmunogenic. Thus, the potential immunogenicity of a γ3immunoadhesin is greater than that of a γ1 immunoadhesin.

NRG3-Ig immunoadhesins are most conveniently constructed by fusing thecDNA sequence encoding the NRG3 portion in-frame to an Ig cDNA sequence.However, fusion to genomic Ig fragments can also be used (see, e.g.Gascoigne et al. (1987) Proc. Natl. Acad. Sci. USA 84:2936-2940; Aruffoet al. (1990) Cell 61:1303-1313; Stamenkovic et al. (1991) Cell66:1133-1144). The latter type of fusion requires the presence of Igregulatory sequences for expression. cDNAs encoding IgG heavy-chainconstant regions can be isolated based on published sequence from cDNAlibraries derived from spleen or peripheral blood lymphocytes, byhybridization or by polymerase chain reaction (PCR) techniques.

Other derivatives of the novel NRG3s of the present invention, whichpossess a longer half-life than the native molecules comprise the NRG3,NRG3 fragment (such as the EGF-like domain) or a NRG3-immunoglobulinchimera, covalently bonded to a nonproteinaceous polymer. Thenonproteinaceous polymer ordinarily is a hydrophilic synthetic polymer,i.e., a polymer not otherwise found in nature. However, polymers whichexist in nature and are produced by recombinant or in vitro methods areuseful, as are polymers which are isolated from native sources.Hydrophilic polyvinyl polymers fall within the scope of this invention,e.g. polyvinylalcohol and polyvinylpyrrolidone. Particularly useful arepolyalkylene ethers such as polyethylene glycol (PEG); polyelkylenessuch as polyoxyethylene, polyoxypropylene, and block copolymers ofpolyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates;carbomers; branched or unbranched polysaccharides which comprise thesaccharide monomers D-mannose, D- and L-galactose, fucose, fructose,D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonicacid, D-mannuronic acid (e.g. polymannuronic acid, or alginic acid),D-glucosamine, D-galactosamine, D-glucose and neuraminic acid includinghomopolysaccharides and heteropolysaccharides such as lactose,amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate,dextran, dextrins, glycogen, or the polysaccharide subunit of acidmucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcoholssuch as polysorbitol and polymannitol; heparin or heparon. The polymerprior to cross-linking need not be, but preferably is, water soluble,but the final conjugate must be water soluble. In addition, the polymershould not be highly immunogenic in the conjugate form, nor should itpossess viscosity that is incompatible with intravenous infusion orinjection if it is intended to be administered by such routes.

Preferably the polymer contains only a single group which is reactive.This helps to avoid cross-linking of protein molecules. However, it iswithin the scope herein to optimize reaction conditions to reducecross-linking, or to purify the reaction products through gel filtrationor chromatographic sieves to recover substantially homogenousderivatives.

The molecular weight of the polymer may desirably range from about 100to 500,000, and preferably is from about 1,000 to 20,000. The molecularweight chosen will depend upon the nature of the polymer and the degreeof substitution. In general, the greater the hydrophilicity of thepolymer and the greater the degree of substitution, the lower themolecular weight that can be employed. Optimal molecular weights will bedetermined by routine experimentation.

The polymer generally is covalently linked to the novel NRG3, NRG3fragment or to the NRG3-immunoglobulin chimeras through amultifunctional crosslinking agent which reacts with the polymer and oneor more amino acid or sugar residues of the NRG3 or NRG3-immunoglobulinchimera to be linked. However, it is within the scope of the inventionto directly crosslink the polymer by reacting a derivatized polymer withthe hybrid, or vice versa.

The covalent crosslinking site on the NRG3 or NRG3-Ig includes theN-terminal amino group and epsilon amino groups found on lysineresidues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxylor other hydrophilic groups. The polymer may be covalently bondeddirectly to the hybrid without the use of a multifunctional (ordinarilybifunctional) crosslinking agent. Covalent binding to amino groups isaccomplished by known chemistries based upon cyanuric chloride, carbonyldiimidazole, aldehyde reactive groups (PEG alkoxide plus diethyl acetalof bromoacetaldehyde; PEG plus DMSO and acetic anhydride, or PEGchloride plus the phenoxide of 4-hydroxybenzaldehyde, succinimidylactive esters, activated dithiocarbonate PEG,2,4,5-trichlorophenylcloroformate or P-nitrophenylcloroformate activatedPEG.) Carboxyl groups are derivatized by coupling PEG-amine usingcarbodiimide.

Polymers are conjugated to oligosaccharide groups by oxidation usingchemicals, e.g. metaperiodate, or enzymes, e.g. glucose or galactoseoxidase, (either of which produces the aldehyde derivative of thecarbohydrate), followed by reaction with hydrazide or amino derivatizedpolymers, in the same fashion as is described by Heitzmann et al. (1974)P.N.A.S. 71:3537-41 or Bayer et al. (1979) Methods in Enzymology 62:310,for the labeling of oligosaccharides with biotin or avidin. Further,other chemical or enzymatic methods which have been used heretofore tolink oligosaccharides are particularly advantageous because, in general,there are fewer substitutions than amino acid sites for derivatization,and the oligosaccharide products thus will be more homogenous. Theoligosaccharide substituents also are optionally modified by enzymedigestion to remove sugars, e.g. by neuraminidase digestion, prior topolymer derivatization.

The polymer will bear a group which is directly reactive with an aminoacid side chain, or the N- or C-terminus of the polypeptide linked, orwhich is reactive with the multifunctional cross-linking agent. Ingeneral, polymers bearing such reactive groups are known for thepreparation of immobilized proteins. In order to use such chemistrieshere, one should employ a water soluble polymer otherwise derivatized inthe same fashion as insoluble polymers heretofore employed for proteinimmobilization. Cyanogen bromide activation is a particularly usefulprocedure to employ in crosslinking polysaccharides.

“Water soluble” in reference to the starting polymer means that thepolymer or its reactive intermediate used for conjugation issufficiently water soluble to participate in a derivatization reaction.“Water soluble” in reference to the polymer conjugate means that theconjugate is soluble in physiological fluids such as blood.

The degree of substitution with such a polymer will vary depending uponthe number of reactive sites on the protein, whether all or a fragmentof the protein is used, whether the protein is a fusion with aheterologous protein (e.g. a NRG3-immunoglobulin chimera), the molecularweight, hydrophilicity and other characteristics of the polymer, and theparticular protein derivatization sites chosen. In general, theconjugate contains about from 1 to 10 polymer molecules, while anyheterologous sequence may be substituted with an essentially unlimitednumber of polymer molecules so long as the desired activity is notsignificantly adversely affected. The optimal degree of cross-linking iseasily determined by an experimental matrix in which the time,temperature and other reaction conditions are varied to change thedegree of substitution, after which the ability of the conjugates tofunction in the desired fashion is determined.

The polymer, e.g. PEG, is cross-linked by a wide variety of methodsknown per se for the covalent modification of proteins withnonproteinaceous polymers such as PEG. Certain of these methods,however, are not preferred for the purposes herein. Cyanuronic chloridechemistry leads to many side reactions, including protein cross-linking.In addition, it may be particularly likely to lead to inactivation ofproteins containing sulfhydryl groups. Carbonyl diimidazole chemistry(Beauchamp et al. (1983) Anal Biochem. 131:25-33) requires high pH(>8.5), which can inactivate proteins. Moreover, since the “activatedPEG” intermediate can react with water, a very large molar excess of“activated PEG” over protein is required. The high concentrations of PEGrequired for the carbonyl diimidazole chemistry also led to problems inpurification, as both gel filtration chromatography and hydrophilicinteraction chromatography are adversely affected. In addition, the highconcentrations of “activated PEG” may precipitate protein, a problemthat per se has been noted previously (Davis, U.S. Pat. No. 4,179,337).On the other hand, aldehyde chemistry (Royer, U.S. Pat. No. 4,002,531)is more efficient since it requires only a 40-fold molar excess of PEGand a 1-2 hr incubation. However, the manganese dioxide suggested byRoyer for preparation of the PEG aldehyde is problematic “because of thepronounced tendency of PEG to form complexes with metal-based oxidizingagents” (Harris et al. (1984) J. Polym. Sci. Polym. Chem. Ed.22:341-52). The use of a Moffatt oxidation, utilizing DMSO and aceticanhydride, obviates this problem. In addition, the sodium borohydridesuggested by Royer must be used at high pH and has a significanttendency to reduce disulfide bonds. In contrast, sodiumcyanoborohydride, which is effective at neutral pH and has very littletendency to reduce disulfide bonds is preferred.

The long half-life conjugates of this invention are separated from theunreacted starting materials by gel filtration. Heterologous species ofthe conjugates are purified from one another in the same fashion. Thepolymer also may be water-insoluble, as a hydrophilic gel.

The novel NRG3s may be entrapped in microcapsules prepared, for example,by coacervation techniques or by interfacial polymerization, incolloidal drug delivery systems (e.g. liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th Edition, Osol, A., Ed. (1980).

H. Antibody Preparation.

(i) Polyclonal Antibodies

Polyclonal antibodies to a NRG3, or fragment thereof (such as theEGF-like domain) of the present invention generally are raised inanimals by multiple subcutaneous (sc) or intraperitoneal (ip) injectionsof the NRG3 and an adjuvant. It may be useful to conjugate the NRG3 or afragment containing the target amino acid sequence to a protein that isimmunogenic in the species to be immunized, e.g. keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for examplemaleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivativesby combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freud's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with ⅕ to {fraction (1/10)} the original amount ofconjugate in Freud's complete adjuvant by subcutaneous injection atmultiple sites. 7 to 14 days later the animals are bled and the serum isassayed for anti-NRG3 antibody titer. Animals are boosted until thetiter plateaus. Preferably, the animal boosted with the conjugate of thesame NRG3, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are used to enhance the immune response.

(ii) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies. For example, the anti-NRG3 monoclonalantibodies of the invention may be made using the hybridoma method firstdescribed by Kohler and Milstein (1975) Nature 256:495, or may be madeby recombinant DNA methods (Cabilly, et al., U.S. Pat. No. 4,816,567).

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,Morrison, et al. (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalentlyjoining to the immunoglobulin coding sequence all or, part of the codingsequence for a non-immunoglobulin polypeptide. In that manner,“chimeric” or “hybrid” antibodies are prepared that have the bindingspecificity of a NRG3 monoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for a NRG3 andanother antigen-combining site having specificity for a differentantigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; biotin; radioactive isotopic labels, such as,e.g., ¹²⁵I, ³²P, ¹⁴C, or ³H, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter, et al. (1962) Nature 144:945; David, et al. (1974)Biochemistry 13: 1014; Pain, et al. (1981) J. Immunol. Meth. 40:219; andNygren (1982) J. Histochem. and Cytochem. 30:407.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and inmmunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

(iii) Humanized Antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al. (1986) Nature 321:522-525; Riechmann et a. (1988) Nature332:323-327; Verhoeyen et a. (1988) Science 239:1534-1536), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (Cabilly, supra), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails see PCT/US93/07832, which is a continuation-in-part ofPCT/US92/05126, which references are herein incorporated by reference intheir entirety.

Alternatively, it is now possible to produce transgenic animals (e.g.mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g. Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA90:2551-255; Jakobovits et al. (1993) Nature 362:255-258.

(iv) Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is for aNRG3 of the present invention the other one is for any other antigen,for example, another member of the NRG3 family. Such constructs can alsobe referred to as bispecific immunoadhesins.

Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello (1983) Nature 305:537-539). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of 10 different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule, which is usually done by affinitychromatography steps, is rather cumbersome, and the product yields arelow. Similar procedures are disclosed in PCT application publication No.WO 93/08829 (published 13 May 1993), and in Traunecker et al. (1991)EMBO 10:3655-3659. This problem may be overcome by selecting a commonlight chain for each arm o the bispecific antibody such that bindingspecificity of each antibody is maintained, as disclosed in U.S.application Ser. No. 08/850058, filed May 5, 1997.

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, and second and thirdconstant regions of an immunoglobulin heavy chain (CH2 and CH3). It ispreferred to have the first heavy chain constant region (CH1) containingthe site necessary for light chain binding, present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are cotransfected into a suitable host organism.This provides for great flexibility in adjusting the mutual proportionsof the three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance. In a preferred embodiment of this approach, the bispecificantibodies are composed of a hybrid immunoglobulin heavy chain with afirst binding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in PCT applicationWO 94/04690 published 3 Mar. 1994.

For further details of generating bispecific antibodies see, forexample, Suresh et al. (1986) Methods in Enzymology 121:210.

(v) Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

I. Diagnostic Kits and Articles of Manufacture.

Since the invention provides a diagnostic assay (i.e. for detectingneurological disorders and for detecting the presence of NRG3 in asample using antibodies or DNA markers) as a matter of convenience, thereagents for these assays can be provided in a kit, i.e., a packagedcombination of reagents, for combination with the sample to be tested.The components of the kit will normally be provided in predeterminedratios. Thus, a kit may comprise the antibody or NRG3 (DNA orpolypeptide or fragment thereof) labeled directly or indirectly with asuitable label. Where the detectable label is an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g. asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers and the like. The relative amounts of the variousreagents may be varied widely to provide for concentrations in solutionof the reagents which substantially optimize the sensitivity of theassay. Particularly, the reagents may be provided as dry powders,usually lyophilized, including excipients which on dissolution willprovide a reagent solution having the appropriate concentration. The kitalso suitably includes instructions for carrying out the bioassay.

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the neurologicaldisorders described herein is provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for treating thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). The active agent in the compositionis NRG3 or an agonist or antagonist thereof. The label on, or associatedwith, the container indicates that the composition is used for treatingthe condition of choice. The article of manufacture may further comprisea second container comprising a pharmaceutically-acceptable buffer, suchas phosphate-buffered saline, Ringer's solution and dextrose solution.It may further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

J. Peptide and Non-Peptide Analogs.

Peptide analogs of the NRG3s of the present invention are modeled basedupon the three-dimensional structure of the native polypeptides.Peptides may be synthesized by well known techniques such as thesolid-phase synthetic techniques initially described in Merrifield(1963) J. Am. Chem. Soc. 15:2149-2154. Other peptide synthesistechniques are, for examples, described in Bodanszky et al., PeptideSynthesis, John Wiley & Sons, 2nd Ed., 1976, as well as in otherreference books readily available for those skilled in the art. Asummary of peptide synthesis techniques may be found in Stuart andYoung, Solid Phase Peptide Synthelia, Pierce Chemical Company, Rockford,Ill. (1984). Peptides may also be prepared by recombinant DNAtechnology, using a DNA sequence encoding the desired peptide.

In addition to peptide analogs, the present invention also contemplatesnon-peptide (e.g. organic) compounds which display substantially thesame surface as the peptide analogs of the present invention, andtherefore interact with other molecules in a similar fashion.

K. Uses of the NRG3s.

Amino acid sequence variants of the native NRG3s of the presentinvention may be employed therapeutically to compete with the normalbinding of the native proteins to their receptor, ErbB4. The NRG3 aminoacid sequence variants are, therefore, useful as competitive inhibitorsof the biological activity of native NRG3s.

Native NRG3s and their amino acid sequence variants are useful in theidentification and purification of the native ErbB4 receptor. Thepurification is preferably performed by immunoadhesins comprising a NRG3amino acid sequence retaining the qualitative ability of a native NRG3of the present invention to recognize its native ErbB4 receptor.

The native NRG3s of the present invention are further useful asmolecular markers of the tissues in which the ErbB4 receptor isexpressed.

Furthermore, the NRG3s, preferably the EGF-like domain of the NRG3 ofthe present invention, provide valuable sequence motifs which can beinserted or substituted into other native members of the NRG3 family ofmolecules, such as the heregulins. The alteration of these nativeproteins by the substitution or insertion of sequences from the novelNRG3s of the present invention can yield variant molecules with alteredbiological properties, such as receptor binding affinity or receptorspecificity. For example, one or more NRG3 domains of another member ofthe NRG3 family may be entirely or partially replaced by NRG3 domainsequences derived from the NRG3s of the present invention. Similarly,EGF-like domain sequences from the NRG3s herein may be substituted orinserted into the amino acid sequences of other NRG3s.

Nucleic acid encoding the NRG3s of the present invention is also usefulin providing hybridization probes for searching cDNA and genomiclibraries for the coding sequence of other NRG3s.

Additionally, NRG3s of the invention are useful in kits for thediagnosis of disease related to NRG3 and for methods of detecting thepresence or absence of NRG3 in a sample, such as a body fluid, asdescribed herein.

Binding and activation of the ErbB4 receptor by NRG3 is expected tomediate such physiological responses in cells expressing the ErbB4receptor as cell growth, cell proliferation, and cell differentiationparticularly in neural tissue. As a result, mammalian NRG3, or an ErbB4receptor binding and activating fragment thereof, is useful in thetreatment of diseases in which neural cell growth, proliferation and/ordifferentiation alleviate symptoms of the disease. The NRG3 may be thefull length amino acid sequence of the murine NRG3 (SEQ ID NO:2) or thehuman NRG3s (SEQ ID NO:6 or SEQ ID NO:23); the full length amino acidsequence from another mammalian species having at least approximately75% homology to the murine and human NRG3 at the amino acid level,preferably about 90% amino acid sequence homology in the EGF-likebinding domain; and an amino acid sequence comprising the EGF-likedomain of NRG3, which sequence binds to the ErbB4 receptor. Where theNRG3 or ErbB4 receptor binding fragment is agonist, the NRG3 or fragmentbinds to and activates ErbB4 receptor. Where the NRG3 or fragment is anantagonist, the NRG3 or fragment binds to but does not activate ErbB4receptor, thereby preventing activation by the naturally occurring NRG3or agonist.

Diseases treatable by administration of NRG3 or an agonist thereof (suchas a polypeptide comprising an NRG3 EGF-like domain) include, but arenot limited to, disorders that may arise in a patient in whom thenervous system has been damaged by, e.g., trauma, surgery, stroke,ischemia, infection, metabolic disease, nutritional deficiency,malignancy, or toxic agents; motoneuron disorders, such as amyotrophiclateral sclerosis (Lou Gehrig's disease), Bell's palsy, and variousconditions involving spinal muscular atrophy, or paralysis; human“neurodegenerative disorders”, such as Alzheimer's disease, Parkinson'sdisease, epilepsy, multiple sclerosis, Huntington's chorea, Down'sSyndrome, nerve deafness, and Meniere's disease; neuropathy, andespecially peripheral, referring to a disorder affecting the peripheralnervous system, most often manifested as one or a combination of motor,sensory, sensorimotor, or autonomic neural dysfunction, such as distalsensorimotor neuropathy, or autonomic neuropathies including reducedmotility of the gastrointestinal tract or atony of the urinary bladder.Examples of neuropathies associated with systemic disease includepost-polio syndrome; examples of hereditary neuropathies includeCharcot-Marie-Tooth disease, Refsum's disease, Abetalipoproteinemia,Tangier disease, Krabbe's disease, Metachromatic leukodystrophy, Fabry'sdisease, and Dejerine-Sottas syndrome; and examples of neuropathiescaused by a toxic agent include those caused by treatment with achemotherapeutic agent such as vincristine, cisplatin, methotrexate, or3′-azido-3′-deoxythymidine. Also, NRG3 or biologically active fragmentsthereof (such as an EGF-like domain of an NRG3) may be used to treatdiseases of skeletal muscle of smooth muscle, such as muscular dystrophyor diseases caused by skeletal or smooth muscle wasting.

Semipermeable, implantable membrane devices are useful as means fordelivering drugs in certain circumstances. For example, cells thatsecrete soluble NRG3, or agonist thereof, or chimeras can beencapsulated, and such devices can be implanted into a patient, forexample, into the brain of patients suffering from Parkinson's Disease.See, U.S. Pat. No. 4,892,538 of Aebischer et al.; U.S. Pat. No.5,011,472 of Aebischer et al.; U.S. Pat. No. 5,106,627 of Aebischer etal.; PCT Application WO 91/10425; PCT Application WO 91/10470; Winn etal. (1991) Exper. Neurology 113:322-329; Aebischer et al. (1991) Exper.Neurology 111:269-275; and Tresco et al. (1992) ASAIO 38:17-23.Accordingly, also included is a method for preventing or treating damageto a nerve or damage to other NRG3-expressing or NRG3-responsive cells,e.g. brain, heart, or kidney cells, as taught herein, which methodcomprises implanting cells that secrete NRG3, or fragment or agonistthereof, or antagonist as may be required for the particular condition,into the body of patients in need thereof. Finally, the presentinvention includes an implantation device, for preventing or treatingnerve damage or damage to other cells as taught herein, containing asemipermeable membrane and a cell that secretes NRG3, or fragment oragonist thereof, (or antagonist as may be required for the particularcondition) encapsulated within the membrane, the membrane beingpermeable to NRG3, or fragment agonist thereof, and impermeable tofactors from the patient detrimental to the cells. The patient's owncells, transformed to produce NRG3 ex vivo, could be implanted directlyinto the patient, optionally without such encapsulation. The methodologyfor the membrane encapsulation of living cells is familiar to those ofordinary skill in the art, and the preparation of the encapsulated cellsand their implantation in patients may be accomplished readily as isknown in the art. The present invention includes, therefore, a methodfor preventing or treating cell damage, preferably nerve damage, byimplanting cells into the body of a patient in need thereof, the cellseither selected for their natural ability to generate NRG3, or fragmentor agonist thereof, or engineered to secrete NRG3, or fragment oragonist thereof. Preferably, the secreted NRG3 is soluble, human NRG3when the patient is human. The implants are preferably non-immunogenicand/or prevent immunogenic implanted cells from being recognized by theimmune system. For CNS delivery, a preferred location for the implant isthe cerebral spinal fluid of the spinal cord.

The administration of the NRG3, fragment or variant thereof, of thepresent invention can be done in a variety of ways, e.g., those routesknown for specific indications, including, but not limited to, orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraarterially, intralesionally,intraventricularly in the brain, or intraocularly. The NRG3 may beadministered continuously by infusion into the fluid reservoirs of theCNS, although bolus injection is acceptable, using techniques well knownin the art, such as pumps or implantation. Sustained release systems canbe used. Where the disorder permits, one may formulate and dose the NRG3variant for site-specific delivery. Administration can be continuous orperiodic. Administration can be accomplished by a constant- orprogrammable-flow implantable pump or by periodic injections.

Semipermeable, implantable membrane devices are useful as means fordelivering drugs in certain circumstances. For example, cells thatsecrete soluble NGF variant can be encapsulated, and such devices can beimplanted into a patient, for example, into the brain or spinal chord(CSF) of a patient suffering from Parkinson's Disease. See, U.S. Pat.No. 4,892,538 of Aebischer et al.; U.S. Pat. No. 5,011,472 of Aebischeret al.; U.S. Pat. No. 5,106,627 of Aebischer et al.; PCT Application WO91/10425; PCT Application WO 91/10470; Winn et al. (1991) Exper.Neurology 113:322-329; Aebischer et al. (1991) Exper. Neurology111:269-275; and Tresco et al. (1992) ASAIO 38:17-23. Finally, thepresent invention includes an implantation device, for preventing ortreating nerve damage or damage to other cells as taught herein,containing a semipermeable membrane and a cell that secretes an NRG3,the cell being encapsulated within the membrane, and the membrane beingpermeable to NRG3, but impermeable to factors from the patientdetrimental to the cells. The patient's own cells, transformed toproduce NRG3 ex vivo, optionally could be implanted directly into thepatient without such encapsulation. The methodology for the membraneencapsulation of living cells is familiar to those of ordinary skill inthe art, and the preparation of the encapsulated cells and theirimplantation in patients may be accomplished readily as is known in theart. Preferably, the secreted NRG3, fragment or variant thereof, is ahuman NRG3 when the patient is human. The implants are preferablynon-immunogenic and/or prevent immunogenic implanted cells from beingrecognized by the immune system. For CNS delivery, a preferred locationfor the implant is the cerebral spinal fluid of the spinal cord.

The pharmaceutical compositions of the present invention comprise a NRG3in a form suitable for administration to a patient. In the preferredembodiment, the pharmaceutical compositions are in a water soluble form,and may include such physiologically acceptable materials as carriers,excipients, stabilizers, buffers, salts, antioxidants, hydrophilicpolymers, amino acids, carbohydrates, ionic or nonionic surfactants, andpolyethylene or propylene glycol. The NRG3 may be in a time-release formfor implantation, or may be entrapped in microcapsules using techniqueswell known in the art.

An effective amount of NRG3 or NRG3 agonist or antagonist to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 10 ng/kg to up to 100 mg/kg of patient body weight or more perday, preferably about 1 μg/kg/day to 10 mg/kg/day. Typically, theclinician will administer NRG3 or NRG3 agonist or antagonist until adosage is reached that achieves the desired effect for treatment of theabove mentioned disorders.

L. Transgenic and Knockout Animals

Nucleic acids which encode novel NRG3 from non-human species, such asthe murine NRG3, can be used to generate either transgenic animals or“knock out” animals which, in turn, are useful in the development andscreening of therapeutically useful reagents. A transgenic animal (e.g.,a mouse) is an animal having cells that contain a transgene, whichtransgene was introduced into the animal or an ancestor of the animal ata prenatal, e.g., an embryonic stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, murine cDNA encoding NRG3 or an appropriatesequence thereof can be used to clone genomic DNA encoding NRG3 inaccordance with established techniques and the genomic sequences used togenerate transgenic animals that contain-cells which express DNAencoding NRG3. Methods for generating transgenic animals, particularlyanimals such as mice, have become conventional in the art and aredescribed, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells, such as neuronal cells, would be targetedfor NRG3 transgene incorporation with tissue-specific enhancers, whichcould result in altered cell differentiation, cell proliferation, orcellular apoptosis, depending upon the ligand interaction with theexpressed polypeptide. Transgenic animals that include a copy of atransgene encoding NRG3 introduced into the germ line of the animal atan embryonic stage can be used to examine the effect of increasedexpression of DNA encoding NRG3. Such animals can be used as testeranimals for reagents thought to confer protection from, for example,diseases associated with abnormal neuronal differentiation and neuronalcell proliferation, for example. In accordance with this facet of theinvention, an animal is treated with the reagent and a reduced incidenceof the disease, compared to untreated animals bearing the transgene,would indicate a potential therapeutic intervention for the disease.

Alternatively, the non-human homologues of NRG3 can be used to constructa NRG3 “knock out” animal which has a defective or altered gene encodingNRG3 as a result of homologous recombination between the endogenous geneencoding NRG3 and altered genomic DNA encoding NRG3 introduced into anembryonic cell of the animal. For example, murine cDNA encoding NRG3 canbe used to clone genomic DNA encoding NRG3 in accordance withestablished techniques. A portion of the genomic DNA encoding NRG3 canbe deleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector (see e.g., Thomas and Capecchi, Cell 51:503(1987) for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected (see, e.g., Li et al.,Cell 69: 915 (1992)). The selected cells are then injected into ablastocyst of an animal (e.g, a mouse) to form aggregation chimeras(see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can be used inthe selection of potential therapeutic agents, such as NRG3 agonists,that restore the cellular processes initiated or maintained by nativeNRG3; or the knockout animals can be used in the study of the effects ofnrg3 mutations.

The instant invention is shown and described herein in what isconsidered to be the most practical, and the preferred embodiments. Itis recognized, however, that departures may be made therefrom which arewithin the scope of the invention, and that obvious modifications willoccur to one skilled in the art upon reading this disclosure.

EXAMPLES

The following examples are provided so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake the compounds and compositions of the invention and how to practicethe methods of the invention and are not intended to limit the scope ofwhat the inventors regard as their invention. Efforts have been made toinsure accuracy with respect to numbers used (e.g. amounts, temperature,etc.), but some experimental errors and deviation should be accountedfor. Unless indicated otherwise, parts are parts by weight, temperatureis in degrees C., and pressure is at or near atmospheric.

Example 1 Molecular Cloning of a Mouse and Human Novel NRG3

Novel NRG3 cDNAs were identified using an expressed sequence tag shownbelow: AATTTCTGCCGAAAACTGATTCCATCTTATCGGATCCAACAGACCACTTGGGGATTGAATTCATGGAGAGTGAAGAAGTTTATCAAAGGCAGGTGCTGTCAATTTCATGTATCATCTTTGGAATTGTCATCGTGGGCATGTTCTGTGCAGCATTCTACTTCAAAAGCAAGAAACAAGCTAAACAAATCCAAGAGCAGCTGAAAGTGCCACAAAATGGTAAAAGCTACAGTCTCAAAGCATCCAGCACAATGGCAAAGTCAGAGAACTTGGTGAAGAGCCATGTCCAGCTGCAAAATAAAATGTCAGGCTTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTNGACCTGCACGTGCACATCCNGATGGCCCGTTTCCTGCCTTTTNTGATGACATTTNCACCACAAATGNAGTGAAAATGGGNCTTTTCNTGCCTTAACTGGTTGACNTTTTTNCCCCAAAAGGAG(EST; SEQ ID NO:21; Genbank entry H23651) from the National Center forBiotechnology Information (NCBI) database of ESTs. This EST from a humanbrain cDNA library, encodes an amino acid sequence having approximately62% identity to amino acids 232-316 of heregulin-β1 (also designatedneuregulin-β1, or NRG 1).

To obtain a partial human cDNA clone, a 50-base single strandedoligonucleotide probe(5′-TGGTAAAAGCTACAGTCTCAAAGCATCCAGCACAATGGCAAAGTCAGAGA-3′; SEQ ID NO:18) was synthesized based on the EST sequence. The probe was used toscreen 1.5×10⁶ plaques from a λgt10 cDNA library prepared from humanfetal brain RNA (HL3003a, Clontech) as described by Godowski et al.(Godowski, P. J. et al. (1989) PNAS USA 86:8083-8087, hereinincorporated by reference in its entirety). Nine positive plaques wereobtained and the sequences of both strands of the largest inserts weredetermined by standard sequencing techniques. From these clonedoverlapping sequences, a partial cDNA sequence of the human NRG3 wasobtained.

Additional 5′ human NRG3 sequence was obtained by anchored PCR of humanhippocampus RNA (Clontech). The complete human open reading framenucleic acid sequence deduced from direct sequencing of hNRG3B1 cDNA isshown in FIG. 2 (SEQ ID NO:5). ATCC 209157 is nucleic acid comprising anexpression vector and the nucleotide sequence of the human NRG3B 1 openreading frame. An alternatively spliced form of human NRG3 was cloned aspRK5.tk.neo.hNRG3B2 (SEQ ID NO:22) encoding the deduced amino acidsequence of SEQ ID NO:23, which amino acid sequence lacks amino acids529 to 552 of SEQ ID NO:6 (see FIG. 4B). Since this alternativelyspliced form of human NRG3 comprises the EGF-like domain of the otherNRG3s as well as high amino acid sequence homology, it is expected toexhibit the biological properties of the NRG3s disclosed herein.

To clone murine NRG3 cDNA sequences, two degenerate primers weredesigned based on regions proximal to the transmembrane domain of thepartial human cDNA, encoding the amino acid sequences NDGECFVI (SEQ IDNO:19) and EFMESEEVY (SEQ ID NO:20). A mouse brain cDNA library(Clontech, ML1042a) was screened, and a clone (C5a) containing a partialmurine NRG3 cDNA was obtained by standard techniques. Using a probederived from the C5a sequence, two additional mouse brain cDNA libraries(ML1034h, Clontech; and 936309, Stratagene) were screened. Both strandsof two overlapping murine partial NRG3 clones, SWAJ-3 and ZAP-1 weresequenced and, together were found to encode an entire open readingframe (ORF) of 2139 bp having the DNA sequence SEQ ID NO:1 and thededuced amino acid sequence SEQ ID NO:2 shown in FIG. 4A. Nucleic acidcomprising the murine NRG3 open reading frame cloned into an expressionvector is designated pLXSN.mNRG3 (ATCC 209156).

The chromosomal localization of human NRG3 was mapped to 10q22 by PCRanalysis of somatic cell hybrid DNA, whereas the NRG1 gene is located at8p11-22 (Lee, J. and Wood, W. I. (1993) Genomics 16:790-791; andOrr-Urtreger, A. et al. (1993) Proc. Natl. Acad. Sci. USA 90:1867-1871).Thus, NRG3 is a novel member of the EGF-like family of protein ligands.

Example 2 Characterization of the Mouse and Human NRG3 Deduced AminoAcid Sequences

The cDNAs of human and murine NRG3 contained open reading framesencoding proteins of 720 and 713 amino acids respectively, withpredicted MW of 77,901 Da for human NRG3 and 77,370 Da for murine NRG3(FIG. 4). The two species of NRG3 are 93% identical in amino acidsequence.

Analysis of the amino acid sequence of human NRG3 revealed that itcontained homology to NRG1 family members (i.e. 23% and 19% sequenceidentity to SMDF (Ho, W. H. et al. (1995) J. Biol. Chem. 270:14523-32)and heregulin-β1 (Holmes, W. E. et al. (1992) Science 256:1205-10)respectively). A hydropathy analysis indicated two hydrophobic segments:W⁶⁶-V⁹¹ and L³⁶²-F³⁸³ (amino acid numbers according to human NRG3).Similar to NPG1, the C-terminal hydrophobic segment may serve as thetransmembrane domain and the N-terminal region may act as internalsignal sequence (Wickner, W. T. and Lodish, H. F. (1985) ScienceM:400-7; Sabatini, D. D. et al. (1982) J. Cell Biol. 92:1-22; andBlobel, G. (1980) Proc. Natl. Acad. Sci. USA 77:1496-500). In contrastto many neuregulin family members, the extracellular domain of NRG3 isdevoid of Ig-like or kringle domains. Instead, NRG3 contains a uniqueAla/Gly rich segment at the N-terminus, a mucin-like SerFThr rich regioncontaining abundant sites for O-linked glycosylation, and an EGF motif.There are no predicted sites for N-linked glycosylation. The EGF-likedomain of NRG3 is distinct from those encoded by the NRG1 (31% identitycompared with neuregulin-β1 EGF-like domain) and NRG2 (39% identity withneuregulin-β1 EGF-like domain), suggesting that NRG3 is not analternatively spliced NRG1 isoform. A diagrammatic comparison ofEGF-like domains of EGF family members is shown in FIG. 5. The putativeintracellular domain of NRG3 contains only approximately 13% sequenceidentity to the intracellular domain of NRG1. The EGF-like domains ofthe EGF family members were obtained from the following sources, eachreference herein incorporated by reference in its entirety. Thesequences compared in FIG. 5 include the EGF-like domain of human NRG3(hNRG3.egf; SEQ ID NO:4; disclosed herein); chicken ARIA (cARIA.egf; SEQID NO:9) (Falls, D. L. et al. (1993) Cell 72:801-815), humanamphiregulin (hAR.egf; SEQ ID NO:10) (Plowman, G. D. et al. (1990) Mol.Cell. Biol. 10:1969-81.); human betacellulin (hBTC.egf; SEQ ID NO:11)(Sasada, R. et al. (1993) Biochem. Biophy. Res. Com. 190:1173-9); humanEGF (hEGF.egf; SEQ ID NO:12)(Nagai, M. et al. (1985) Gene 36:183-8.);human heparin-binding EGF-like growth factor (hHB-EGF.egf; SEQ ID NO:13)(Higashiyama, S. et al. (1991) Science 251:936-9.); human heregulin-α(hHRGα; SEQ ID NO: 14); human heregulin-β(hHRGβ.egf; SEQ IDNO:15)(Holmes, W. E. et al. (1992) Science 256:1205-1210); human TGF-α(hTGFα.egf; SEQ ID NO:16) (Derynck, R. et al. (1984) Cell 38:287-97.);and mouse epiregulin (mEPR.egf; SEQ ID NO: 17) (Toyoda, H. et al. (1995)FEBS Lett. 377:403-7.).

Example 3 Expression of Murine and Human NRG3

A. Northern Blot Analysis of Human tissue. The tissue expression profileof the human NRG3 was examined by Northern blot analysis. A multi-tissueRNA blot containing 2 μg each of poly(A)⁺ RNA from human tissues werepurchased from Clontech. The region of the human NRG3 nucleic acidsequence encoding amino acids 394 to 536 was used to generate DNAhybridization probes by PCR amplification. The DNA probes were labeledwith α-³²P-dCTP by random priming (Promega). The RNA blot was hybridizedwith 50% fornamide, 5×SSC, 50 mM potassium phosphate (pH 7.0),5×Denhardt's, 10% dextran sulfate at 42° C. for 20 hr. The blot waswashed with 0.1×SSC, 0.1% SDS at 50° C. for 30 min and exposed inPhosphoImager™. Expression of NRG3 is mixtures of tissues was used as aguide to determine expression in specific tissues by in situhybridization.

B. In situ Hybridization Analysis of Mouse Tissues. Formalin-fixed,paraffin-embedded mouse embryos (embryonic days 13, 14, 16), andglutaraldehyde-fixed, paraffin-embedded or paraformaldehyde-fixed,frozen adult mouse brain, ovary, jejunum, kidney, adrenal, lung,stomach, spleen, skeletal muscle, liver and colon were sectioned andprocessed for in situ hybridization by the method of Lu and Gillett (Lu,L. H. and Gillett, N. A. (1994) Cell Vision 1:169-176) withmodifications. Briefly, the in situ hybridization probe was generated byin vitro transcription directly from a PCR fragment, rather than from aplasmid DNA as described. ³²P-UTP-labeled sense and antisense riboprobeswere generated by labeling PCR products of a cDNA fragment encodingamino acids C²⁹² to N⁴⁸² of murine NRG3.

C. Northern Blot And In Situ Hybridization Analyses Reveal a NeuralExpression Pattern of NRG3. A 4.4 kb mRNA transcript that hybridized tothe probe derived from amino acids 394 to 536 of human NRG3 was highlyexpressed in brain. In a Northern blot of various brain tissues, NRG3expression was detected at high levels in most regions of the brain withthe exception of corpus callosum. A lower level expression of a 1.9-kbtranscript was detected in testis. The 4.4-kb transcript, but not the1.9-kb transcript, is of sufficient size to encode NRG3, suggesting thatthe smaller transcript may encode an alternatively spliced form of NRG3.A similar pattern of expression of NRG3 was observed in RNA blots frommurine tissues using a probe derived from the region of murine NRG3 thatoverlaps the EGF-like domain.

The tissue distribution of NRG3 expression was characterized by in situhybridization using tissues of embryonic and adult mice. At embryonicday 13 (E13) (the earliest time point examined), NRG3 mRNA was confinedto the nervous system. A strong signal for NRG3 mRNA in the brain,spinal cord, trigeminal, vestibular-cochlear and spinal ganglia ofembryonic day 16 (E16) mice was also demonstrated. Regions of thetelencephalon containing differentiating cells (e.g., the corticalplate) displayed an intense NRG3 signal, whereas the underlying regionscontaining proliferating or migrating cells (ventricular andsubventricular zones), showed little expression. Thus, NRG3 appeared tobe expressed mainly in the nervous system of embryonic mice. In adultanimals NRG3 antisense probes hybridized to mRNA in spinal cord andnumerous brain regions including deep cerebellar nuclei, vestibularnuclei, cerebral cortex, piriform cortex, anterior olfactory nucleus,medial habenula, hippocampus, hypothalamus and thalamus.

Example 4 Characterization of the Binding Characteristics of NRG3Fragments

A. Expression and Purification of NRG3^(EGF) Fusion Protein in MammalianCells

To examine the binding characteristics of the NRG3 EGF-like domain aswell as to demonstrate the functionality of an NRG3 fragment of theinvention, a soluble fusion protein was prepared comprising a sequenceof EGF-like domain, which domain has the same amino acid sequence inmouse and human NRG3.

A secreted, epitope tagged polypeptide comprising the EGF-like domain ofmurine NRG3₂₈₄₋₃₄₄ was constructed by linking in the expressedN-terminal to C-terminal direction 1) the coding sequence for the gDsignal sequence and epitope tag (Mark, M. R. et al. (1994) J. Biol.Chem. 269, 10720-10728); 2) the sequences encoding amino acids 284-344of murine NRG3 (identical to human NRG3 amino acids 282 to 342); and 3)the coding sequences of the Fc portion of human IgG₁ in pSAR.SD5 vector(psar.SD5, from A. Shen, Genentech, Inc.). The plasmid encoding thesesequences was designated NRG3^(EGF).Fc. The NRG3^(EGF).Fc expressionplasmid was transfected using LipofectAMINE (GIBCO/BRL, Bethesda, Md.)into DHFR: Chinese hamster ovary cells (CHO/DP12; ATCC designation CCL9096). Clones were selected in glycine/hypoxanthine/thymidine minusmedium see, for example, (Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory (1989)), pooled, andexpanded. The encoded fusion protein was expressed in cultures of theselected clones. Conditioned media from these cells were collected andthe recombinant protein purified by a HiTrap protein A affinity column(Pharmacia).

A monomeric fusion protein designated NRG3^(EGF).H6 fusion protein wasproduced in the same system as the Fc-fusion protein and purifiedthrough a cobalt affinity column. NRG3^(EGF).H6 comprises the N-terminalgD tag, murine NRG3₂₈₄₋₃₄₄, and a coding sequence for six histidineresidues. Purification was based on the affinity of the histidine sidechains for immobilized cobalt using a cobalt affinity column (Cobaltaffinity column, R. Vandlen, Genentech, Inc.). Protein concentration wasdetermined by BioRad Protein Assay (BioRad, Richmond, Calif.).

B. Generation of K562^(erbB) Cell Lines. Stable cell lines thatexpressed human ErbB2, ErbB3 or ErbB4 receptors were derived from K562cells (K562 cells have ATCC designation CCL 243). cDNAs of human erbB2,erbB3 and erbB4 were from L. Bald and G. Scoffer, Genentech (Sliwkowski,M. et al. (1994), J. Biol Chem. 269:14661-14665). These cDNAs weresubcloned into CMV-based expression vectors and introduced into the K562human myeloid leukemia cell line by electroporation (1180 mF, 350 V).The transfectants were cultured in RPMI 1640 supplemented with 10% fetalbovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 mg/mlstreptomycin, and 10 mM HEPES containing 0.8 mg/ml G418. Resistantclones were obtained by limiting dilution, and receptor expression wasconfirmed by western blot and NRG binding assays. Receptor expressionwas confirmed by western blot analysis using antibodies for each of theErbB receptors (antibodies prepared at Genentech, Inc.) Phorbol esterstimulation was found to significantly enhance receptor expression inboth the ErbB3 and ErbB4 transfectants, and the stably transfected K562cell lines were cultured in medium containing 10 ng/ml Phorbol,12-Myristate, 12-Acetate-(PMA) overnight prior to use.

C. FACS Analysis. For each binding reaction, 5×10⁵ stably transfectedK562 cells were suspended in PBS/2% BSA at 4° C. for 30 min followed byincubation with 5 μg of isolated, purified NRG3^(EGF).Fc (MW 90 kDa) ina volume of 0.25 ml on ice for 60 min. 1 μg of primary antibody (anti-gDor anti-ErbB receptor) and secondary PE-conjugated (CALTAG, CA., goatanti-mouse, 1:100 dilution) antibodies were added sequentially with30-60 min incubation time and extensive washes before each addition.FACS analyses were performed on a Becton & Dickson FACS instrument.Anti-gD (5B6), anti-ErbB2 receptor (4D5), anti-ErbB3 receptor (2F9) andanti-ErbB4 receptor (3B9) monoclonal antibodies were prepared usingstandard techniques by the Monoclonal Antibody Group, Genentech, Inc.

D. The EGF-Like domain of NRG3 Binds Specifically to the ErbB4 ReceptorTyrosine Kinase. To identify the receptor(s) for NRG3, the ability ofNRG3 to bind to any of the known neuregulin receptors was investigated.Stable cell lines were generated which expressed receptors ErbB2, ErbB3,or ErbB4. The parental cell line K562 does not express detectable levelsof ErbB receptors (FIG. 6A). K562^(erb2), K562^(erb3) and K562^(erb4)cells expressed only the corresponding receptors (FIGS. 6B-6D).

Since the EGF-like domain determines the binding specificity of NRG1 totheir receptors, a protein containing an epitope tagged version of theEGF-like domain of NRG3 fused to the Fc portion of human IgG wasexpressed and purified. Using a FACS assay, it was observed thatNRG3^(EGF).Fc bound to cells expressing ErbB4 receptor (FIG. 6H).Binding was specific in that NRG3^(EGF).Fc did not bind to either theparental K562 cells, or cells expressing either ErbB2 or ErbB3 (FIGS.6E-6G). A control fusion protein, RSE.Fc, did not bind to any of thesecell lines. This binding of NRG3^(EGF).Fc to K562^(erb4) cells wascompeted in a dose-dependent fashion by the EGF-like domain ofheregulin-β1 (NRG1^(EGF)), but not by RSE.Fc, suggesting thatNRG3^(EGF).Fc interacts directly with ErbB4 receptors on the cellsurface.

A soluble form of the ErbB4 receptor was co-immunoprecipitated byNRG3^(EGF).Fc in vitro, further demonstrating the binding ofNRG3^(EGF).Fc to ErbB4 receptor.

The binding of NRG3^(EGF).Fc to ErbB4 receptor was further analyzed bydirect competitive binding assays using ¹²⁵I-labeled NRG3^(EGF).Fc.Purified NRG3^(EGF).Fc was radio-iodinated using the lactoperoxidasemethod as described by Sliwkowski et al. (Sliwkowski, M. X. et al.(1994) J. Biol. Chem. 269, 14661-5). The average specific activity ofthe radiolabeled protein was 300 μCi/μg. Binding of ¹²⁵I-NRG3^(EGF).Fcto immobilized ErbB4.Fc was competed by either NRG3^(EGF).Fc or EGFdomain of NRG1^(EGF) (rHRGβ1₁₇₇₋₂₄₄) in a concentration dependentmanner.

The displacement binding assays were performed in Maxisorp C 96-wells(Nunc, Naperville, Ill.). Goat anti-human antibody (Boehringer Mannheim,Germany) was coated on the plate at a concentration of 0.2 μg/well in100 μl of 50 mM sodium carbonate buffer (pH 9.6) at 4° C., overnight.The plate was blocked by 1% BSA in TBST buffer (25 mM Tris, pH 7.5, 150mM NaCl, 0.02% Tween 20) for 30 min at room temperature (RT). A solubleform of ErbB4 receptor was added at 15 ng/well in 1% BSA/TBST andincubated for 1.5 hr at RT. To prevent radiolabeled protein frominteracting with residual goat anti-human antibodies, 1 μM of ahumanized monoclonal antibody (rhuMAB HER2; Carter, P. et al. (1992)Proc. Natl. Acad. Sci. USA 89:4285-9) was added to the plate for 20 minand was included in the subsequent binding reaction.

The competitive binding assay was then initiated by the addition of 80pM (200,000 cpm) of ¹²⁵I-NRG3^(EGF).Fc along with various concentrationsof unlabeled NRG3^(EGF).Fc or NRG1^(EGF) (E. coli-expressed, withoutFc). NRG1^(EGF) is the EGF domain of NRG1, corresponding to amino acids177-244 of the neuregulin-β1 isoform (Holmes, W. E. et al. (1992)Science 256:1205-10) and obtained from J. A. Lofgren, Genentech, Inc.The final incubation volume was 100 μl in binding buffer (F-12/DMEMmedium, 50 mM HEPES, pH7.5, 2% BSA) and the reaction was allowed toproceed at RT for 1.5 hr. The unbound material was washed by TBSTextensively, and the bound radioactivity was counted on a BeckmanIsoData gamma-counter (Smith-Kline Beckman, PA). Data was analyzed usinga nonlinear regression computer program.

Based on the results of the binding experiments as shown in FIGS. 6A-6H,the estimated affinity (K_(i)) for NRG3^(EGF).Fc for binding to ErbB4.Fcwas determined to be 9±4 nM (n=4), and the apparent K_(i) of NRG1^(EGF)was approximately 1 nM. The shallowness of the displacement curve ofNRG3^(EGF).Fc may be due to the fact that the NRG3^(EGF).Fc is expressedas a bivalent Fc fusion protein (FIG. 7). The results of the controlexperiments showed that ¹²⁵I-NRG3^(EGF).Fc did not bind control receptorRSE.Fc in the same experiment, and RSE.Fc did not compete¹²⁵I-NRG3^(EGF).Fc bound to ErbB4.Fc.

E. Tyrosine Phosphorylation Assay. NRG1 binds and activates ErbB2, ErbB3and ErbB4 receptor resulting in tyrosine phosphorylation and downstreamsignaling events (Sliwkowski, M. X., et al. (1994), supra; Plowman, G.D. et al. (1993) supra; and Carraway, K. L. and Cantley, L. C. (1994),surpa). As demonstrated in the preceding example, NRG3 binds ErbB4receptor, but not ErbB2 or ErbB3 receptors at a detectable level. Theability of the EGF-like domain of NRG3 (NRG₃ ^(EGF)) to activate ErbB4receptor, K562^(erbB4) cells was examined.

K562^(erbB4) cells or MDA-MB-453 cells (negative control; ATCCdesignation HB 131) were cultured in medium lacking serum for 12 hoursand then stimulated with NRG3^(EGF).Fc, NRG^(EGF).H6 or NRG1^(EGF).K562^(erbB4) cells were treated with 2.5 nM or 25 nM of NRG3^(EGF).Fcfor 3 min or 8 min. As a positive control, the cells were similarlytreated with NRG1^(EGF).

ErbB4 receptor tyrosine phosphorylation was detected byimmunoprecipitation and Western blot according to the followingprocedure. Cells were lysed with lysis buffer (20 mM Tris, pH 7.5, 100mM NaCl, 30 mM NaF, 2 mM EDTA, 2 mM EGTA, 0.1% SDS, 1% Triton X-100, 2mM sodium vanadate, 2 mM sodium molybdate, 2 mM of PMSF). After removingcell debris by centrifugaton, 1 μg of anti-ErbB4 receptor monoclonalantibody (C-18, Santa Cruz Biotechnology, Santa Cruz, Calif.) was addedtogether with 20 μl of protein A-agarose slurry (Sigma, St. Louis, Mo.).Immunoprecipitation was performed at 4° C. overnight, complexes werecollected by centrifugation and washed three times with 1 ml lysisbuffer. Proteins were separated by reducing SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) on Novex 4%-12% minigels and transferred tonitrocellulose. The blots were probed with peroxidase conjugatedanti-phosphotyrosine antibody (Transduction Laboratory). The blot wasstripped and reprobed with anti-ErbB4 receptor antibody followed byperoxidase conjugated goat anti-rabbit IgG antibody (Sigma) to visualizeErbB4 receptor proteins.

Based on these experiments, it was demonstrated that NRG3^(EGF).Fcstimulated ErbB4 receptor tyrosine phosphorylation at both time pointsand in a dose dependent manner.

To confirm the ability of NRG3^(EGF) to activate the ErbB4 receptortyrosine phosphorylation, receptor activation in the human breast cancercell line MDA-MB-453 was examined. This cell line expresses high levelof ErbB2 and ErbB3 receptors, and low levels of ErbB4 receptor.Treatment of MDA-MB-453 cells with NRG3^(EGF).Fc or with a monomericform of the EGF domain (NRG3^(EGF).H6) resulted in substantial increaseof tyrosine phosphorylation of ErbB4 receptor.

NRG family members and other members in the EGF family display a complexpattern of receptor binding. In most cases, one ligand is able to bindseveral combinations of receptor homo- and heterodimers (Karunagaran, D.et al. (1996) EMBO J 15:254-264,Beerli, R. R. and Hynes, N. E. (1996) J.Biol. Chem. 271:6071-6076). For example, NRGs bind ErbB2/ErbB3 receptorheterodimers and ErbB4/ErbB4 receptor homodimers with high affinity butErbB3/ErbB3 receptor homodimers with low affinity (Sliwkowski, M. X. etal. (1994) J. Biol. Chem. 269, 14661-5, Carraway, K. L. and Cantley, L.C. (1994) Cell 78, 5-8, Tzahar, E. et al. (1994) J. Biol. Chem. 269,25226-33, Carraway, K. L. r. et al. (1994) J. Biol. Chem. 269, 14303-6,and Kita, Y. A. et al. (1994) FEBS Lett. 349, 139-43). Betacellulinbinds both EGFR and ErbB4 homodimers (Riese, D. J. et al. (1995) Mol.Cell. Biol. 15:5770-6). The EGF-like domains of EGF and NRG1 familymembers determine the specificity of receptor activation (Barbacci, E.G. et al. (1995) J. Biol. Chem. 270:9585-9589). The limited amino acidsequence homology in the EGF-like domains of NRG3 and NRG1 suggests thatNRG3 may have a different spectrum of receptor interactions relative tomembers of the NRG family, but with potentially overlapping bindingsites, since binding of the EGF-like domain of NRG3 to ErbB4 can becompeted by the EGF-like domain of NRG1.

NRG3^(EGF).Fc did not bind to K562 cells that express either ErbB2 orErbB3 (FIGS. 6E-6G), or to MDA-MB-486 cells which express high levels ofthe EGFR. An increase in phosphorylation of either the EGFR, ErbB2 orErbB3 in MDA-MB-453 cells treated with NRG3 also was not observed.

Most variants of NRGs, with the exception of the neural specific form ofSMDF, are widely expressed in numerous tissues including brain, heart,skeletal muscle, breast, liver, lung, among others. Betacellulin, aligand for both EGFR and ErbB4, also displays broad tissue expressionpatterns (Shing, Y. et al. (1993) Science 259, 1604-7; Sasada, R. et al.(1993) Biochem. Biophy. Res. Com. 190, 1173-9). In contrast, theexpression of NRG3 is strikingly restricted to neural tissues asdisclosed herein by Northern analysis and in situ hybridization.Developmentally, NRG3 mRNA can be detected as early as E11 (but not E4)in mouse as judged by Northern blot and E13 by in situ hybridization(the earliest age examined). ErbB4 is predominantly expressed in brain,heart and skeletal muscle (Plowman, G. D. et al. (1993) Proc. Natl.Acad. Sci. USA 90, 1746-50). ErbB4 was also shown to be broadlydistributed in the brains of chick embryos (E14, E17, predominantly inneurons) (Francoeur, J. R. et al. (1995) J. Neur. Res. 41, 836-45), inrat retina cultures (Bermingham-McDonogh, O. et al. (1996) Development122, 1427-38.), at neuromuscular synapses (Zhu, X. et al.(1995) EMBO J.14, 5842-8.), but not in cultured human and rat Schwann cells (Grinspan,J. B. et al. (1996) J. Neuroscience 16, 6107-6118,Levi, A. D. et al.(1995) J. Neuroscience 15, 1329-40.). Recently, ErbB4 was found toco-localize with GABA⁺ cells (Weber, J. et al. (1996) Soc Neurosci Abstr22, 1579.). Thus, the same receptor may mediate distinct biologicalfunctions in different tissues or cell types when interacted withcorresponding tissue-specific ligands. For example, NRG1 may serve as aligand for ErbB4 during heart development, betacellulin may act as amitogenic ligand for ErbB4 in variety of cell types, while neuralspecific ligand(s) (such as NRG3) may function as trophic or guidancemolecules on ErbB4 receptor expressing cells in the central orperipheral nervous systems.

Example 5 Binding and ErbB4 Receptor Tyrosine Kinase Activation by FullLength Mouse and Human NRG3s

A full length murine NRG3 or human NRG3 was synthesized based on themurine and human consensus nucleic acid sequences SEQ ID NO:1 and SEQ IDNO:5, respectively and the NRG3s were expressed as amino acid sequences.Based on the experiments described herein for the characterization ofNRG3-EGF binding and ErbB4 receptor activation, analogous experimentsare performed for the full length consensus NRG3 from mouse and humansources. Adjustments to the reaction conditions are made to optimize pH,solutes and their concentrations, and other relevant parameters to allowErbB4 receptor-binding of the full length consensus NRG3 and ErbB4receptor activation.

Alternatively, a murine or human NRG3 polypeptide fragment comprisingthe EGF-like domain but lacking the transmembrane domain is synthesizedand tested for ErbB4 receptor binding and activation as describedherein. Such a NRG3 fragment may, for example, include the extracellulardomain of a NRG3, which extracellular domain contains the EGF-likedomain.

A NRG3 extracellular domain may optionally be fused to an immunoglobulinconstant region, as shown herein for the NRG3-EGF-Fc fusion proteins. Asan Fc fusion protein, the NRG3 extracellular domain-Fc protein isexpected to form a dimer. The immunoglobulin constant region ispreferably from IgG, but may also be taken from IgM, IgA and IgE andremain within the scope of the invention.

Where a monomeric fusion protein is desired that retains bindingactivity or binding and activation ability, the extracellular domain isfused to, for example, a series of histidine residues as disclosedherein for the NRG3-EGF-H6 immunoadhesion.

Adjustments to the binding reaction conditions are made to optimize pH,solutes and their concentrations and other relevant parameters to allowErbB4 receptor-binding of the NRG3 fragment and ErbB4 receptoractivation.

Example 6 Enhancement of Cellular Proliferation

Enhancement of cellular proliferation is exemplified by the followingassay in which cells expressing ErbB4 receptor on their surface aretreated with NRG3. It is understood that according to the invention, thecells may be treated with a NRG3 fragment (such as the NRG3 EGF-likedomain) or a NRG3 variant.

As an example, rat retina cells which naturally express ErbB4 receptor(Bermingham-McDonogh, O. et al. (1996) Development 122, 1427-38) arecultured by standard techniques. The cultured cells are contacted withNRG3 in a dose dependent manner and an increase in cell number (e.g. a30% percent increase at 48 hours) and EC50 is determined.

NRG3 treatment may also alter the morphology of these cells; untreatedcells were multipolar with numerous branched processes whereasNRG3-treated cells may become spindle-shaped smooth processes and/oralign themselves in a parallel array.

NRG3 is believed to stimulate neuronal cell growth in a dose dependentmanner. NRG3 alone is expected to produce a significant increase inneuronal cell number compared to control medium. A synergistic effectmay be observed between other neuronal proliferation enhancers such asgas6 (growth arrest-specific gene; see, for example, Schneider et al.,Cell 54:787-793 (1988); and Manfioletti et al. in Molec. Cell Biol.13(8):4976-4985 (1993)) and/or heregulin. NRG3 is expected to increaseboth cell number and thymidine incorporation as measures of cellproliferation.

NRG3 is expected to have an effect on cell morphology as determined byviewing phase contrast micrographs of ErbB4 receptor-expressing neuronalcells grown in various media containing NRG3 alone or NRG3 plus othercell proliferation enhancing compounds such as heregulin, gas6, fetalbovine serum, and the like. Photomicrographs are taken after 96 hours ofculture. The cells grown in the presence of NRG3 are expected to haveprocesses which are not observed in cells grown in the absence of NRG3.

Cells are stained by immunofluorescence for markers specific for thecultured neuronal cells. Briefly, passaged ErbB4 receptor-expressingneuronal cells are contacted with NRG3 and cultured for 24 hours onlaminin coated Chamber slides and fixed in 10% formalin in PBS. Fixedcells are blocked with 10% goat serum and incubated with rabbit derivedanti-marker antibody at dilutions recommended by the distributor.Specific binding of the primary antibody is observed by staining withgoat anti-rabbit IgG (Fab′)₂-FITC conjugates. Cells are counter-stainedwith DNA dye propidium iodide. Negative controls are run on WI-38 cellswhich stain negative. Cells grown under these conditions are expected toshow 100% immunofluorescent staining for the cell markers.

The ability of NRG3 to stimulate proliferation in ErbB4receptor-expressing neuronal cells through the ErbB4 tyrosine kinasereceptors may be investigated as follows. Cells are stimulated withvarious amounts of NRG3 (for example, 0 to 200 nM) for 15 min in a 37°C. incubator. Cell lysates are prepared and immunoprecipitated with ananti-ErbB4 receptor antibody. Tyrosine phosphorylation of ErbB4 receptoris detected with 4G10 anti-phosphorylation antibody. Approximately 10⁶cells are grown to near confluence in defined media. Cells are treatedwith NRG3 for 15 min in a 37° C. incubator and lysed on ice with 1 ml oflysis buffer (20 mM HEPES, pH7.4, 135 mM NaCl, 50 mM NaF, 1 mM sodiumvanadate and 1 mM sodium molybdate, 2 mM EDTA and 2 mM EGTA, 10%glycerol, 1% NP40, 1 μM okadaic acid, 1 mM PMSF and 1 mM AEBSF). Celllysates are clarified by centrifuging at 14000×g at 4° C. for 10 min.Immunoprecipitations are performed using approximately 1 μg of rabbitanti-ErbB4 receptor antibody or 2 μl of rabbit anti-ErbB4 receptorantiserum. Immunocomplexes are collected with 10 μl of Protein ASepharose CL-4B beads. Proteins are separated on Novex 4-12% gradientgel and transferred onto nitrocellulose membrane. Anti-phosphotyrosineimmunoblots are performed using 4G10 mouse anti-phosphotyrosine antibody(UBI), goat anti-mouse horseradish peroxidase conjugate and ECLdeveloping kit (Amersham). Addition of NRG3 to ErbB4 receptor-expressingneuronal cells is expected to cause autophosphoralation of ErbB4receptor tyrosine residue(s).

It is beneficial to have populations of mammalian neuronal cells(preferably human cells) for use as cellular prostheses fortransplantation into areas of damaged spinal cord in an effort toinfluence regeneration of interrupted central axons, for assisting inthe repair of peripheral nerve injuries and as alternatives to multipleautografts. See Levi et al., J. Neuroscience 14(3):1309-1319 (1994). Theuse of cell culture techniques to obtain an abundant source ofautologous graft material from a small biopsy has already met withclinical success in providing human epidermal cells to cover extensiveburns (Gallico et al., N. Eng J. Med. 311:338-451 (1984)). Accordingly,it is expected that the above approach will meet with success inmammals, including humans.

All documents cited throughout the specification as well as thereferences cited therein are hereby expressly incorporated by referencein their entirety. While the present invention is illustrated withreference to specific embodiments, the invention is not so limited. Itwill be understood that further modifications and variations arepossible without diverting from the overall concept of the invention.All such modifications are intended to be within the scope of thepresent invention.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):ATCC Material Dep. No. Deposit Date mouse NRG3 pLXSN.mNRG3 209156 Jul.22, 1997 human NRG3B1 pRK5.tk.neo.hNRG3B1 209157 Jul. 22, 1997 humanNRG3B2 pRK5.tk.neo.hNRG3B2 209297 Sep. 23, 1997

These deposits are made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. A polypeptide comprising an amino acid sequence encoding an EGF-likedomain, wherein the amino acid sequence has the binding characteristicsof NRG3.
 2. The polypeptide of claim 1 wherein the bindingcharacteristics of NRG3 comprise (a) binding to ErbB4 receptor but notto ErbB2 receptor or ErbB3 receptor under experimentally comparableconditions; and (b) activation of ErbB4 receptor tyrosinephosphorylation.
 3. The polypeptide of claim 1 wherein the amino acidsequence has at least 75% amino acid sequence homology to the amino acidsequence SEQ ID NO:4.
 4. The polypeptide of claim 1, wherein thepolypeptide binds to the ErbB4 receptor and stimulates tyrosinephosphorylation of the ErbB4 receptor.
 5. A polypeptide that binds ErbB4receptor, which polypeptide is selected from the group consisting of (a)a polypeptide comprising an amino acid sequence having at least 75%sequence homology to the extracellular domain NRG3 (SEQ ID NO:3 or 7).(b) a polypeptide comprising an amino acid sequence having at least 75%sequence homology to SEQ ID NO:2 or SEQ ID NO:6; (c) a further mammalianhomologue of polypeptide (a) or (b): (d) a soluble form of any of thepolypeptides (a)-(c) having a transmembrane domain that cannot anchorthe polypeptide in a cell membrane; and (e) a derivative of any of thepolypeptides (a)-(d) having the binding characteristics of NRG3.
 6. Thepolypeptide of claim 1 encoded by a NRG3 nucleic acid open reading framesequence in ATCC deposit 209156 (pLXSN.mNRG3).
 7. The polypeptide ofclaim 1 encoded by a NRG3 nucleic acid open reading frame sequence inATCC deposit 209157 (pRK5.tk.neo.hNRG3B1).
 8. The polypeptide of claim 1encoded by a NRG3 nucleic acid open reading frame sequence in ATCCdeposit 209297 (pRK5.tk.neo.hNRG3B2).
 9. The polypeptide of claim 1which is devoid of a cytoplasmic domain, or devoid of a transmembranedomain that can anchor the polypeptide in a cell membrane, or both. 10.The polypeptide of claim 1 unaccompanied by native glycosylation. 11.The polypeptide of claim 1 which has a variant glycosylation.
 12. Anantagonist of the polypeptide of claim
 1. 13. An agonist of thepolypeptide of claim
 1. 14. An isolated nucleic acid molecule encodingthe polypeptide of claim
 1. 15. The nucleic acid molecule of claim 14further encoding the extracellular domain of a mammalian NRG3.
 16. Thenucleic acid molecule of claim 15, wherein the encoded extracellulardomain has at least 75% amino acid sequence identity to the amino acidsequence of SEQ ID NO:3 or SEQ ID NO:7.
 17. The nucleic acid molecule ofclaim 14 wherein the encoded amino acid sequence is devoid of acytoplasmic domain or a transmembrane domain that can anchor thepolypeptide in a cell membrane, or both.
 18. An expression vectorcomprising the nucleic acid molecule of claim 14 operably linked tocontrol sequences recognized by a host cell transformed with the vector.19. An expression vector according to claim 18 obtainable as ATCC 209156(pLXSN.mNRG3).
 20. An expression vector according to claim 18 obtainableas ATCC 209157 (pRK5.tk.neo.hNRG3B1).
 21. An expression vector accordingto claim 18 obtainable as ATCC 209297 (pRK5.tk.neo.hNRG3B2).
 22. A hostcell comprising the vector of claim
 18. 23. The host cell of claim 22which is a mammalian cell.
 24. The host cell of claim 23 which is aChinese hamster ovary cell line.
 25. A method for producing the aminoacid sequence encoding an EGF-like domain that binds ErbB4 receptor, themethod comprising: a) culturing a cell comprising the nucleic acid ofclaim 14; and b) recovering the polypeptide from the cell culture. 26.The method of claim 25 wherein the polypeptide is secreted into theculture medium and recovered from the culture medium.
 27. An antibodythat specifically binds to the polypeptide of claim
 1. 28. A hybridomacell line producing the antibody of claim
 27. 29. An immunoadhesincomprising the polypeptide of claim 1 fused to an immunoglobulinsequence.
 30. The immunoadhesin of claim 29, further comprising theEGF-like domain of SEQ ID NO:4.
 31. The immunoadhesin of claim 29wherein the immunoglobulin sequence is an immunoglobulin heavy chainconstant domain sequence.
 32. The immunoadhesin of claim 31 wherein theimmunoglobulin sequence is a constant domain sequence of an IgG-1, IgG-2or IgG-3.
 33. A method of detecting an NRG3 in a sample, the methodcomprising: a) contacting the antibody of claim 27 with the sample; b)detecting binding of the antibody to a polypeptide in the sample,wherein the polypeptide is an NRG3.
 34. A method of detecting ErbB4receptor in a sample, the method comprising: a) contacting thepolypeptide of claim 1 with the sample; and b) detecting binding of theamino acid sequence to a protein in the sample.
 35. The method of claim34 wherein the sample comprises a cell expressing ErB4 receptor on itssurface.
 36. The method of claim 35 wherein the sample is a mammaliantissue sample.
 37. A method of administering a NRG3 polypeptide to amammal experiencing a disorder treatable with NRG3, wherein the methodcomprises introducing into the mammal a cell comprising the nucleic acidof claim 14, and wherein the NRG3 polypeptide is secreted by the cell.38. The method of claim 37 wherein the cell is contained within a porousmatrix and the matrix is administered to the mammal.